Nitric oxide radicals are emitted by wasp eggs to kill mold fungi

Detrimental microbes caused the evolution of a great diversity of antimicrobial defenses in plants and animals. Insects developing underground seem particularly threatened. Here we show that the eggs of a solitary digger wasp, the European beewolf Philanthus triangulum, emit large amounts of gaseous nitric oxide (NO^⋅) to protect themselves and their provisions, paralyzed honeybees, against mold fungi. We provide evidence that a NO-synthase (NOS) is involved in the generation of the extraordinary concentrations of nitrogen radicals in brood cells (~1500 ppm NO^⋅ and its oxidation product NO₂^⋅). Sequencing of the beewolf NOS gene revealed no conspicuous differences to related species. However, due to alternative splicing, the NOS-mRNA in beewolf eggs lacks an exon near the regulatory domain. This preventive external application of high doses of NO^⋅ by wasp eggs represents an evolutionary key innovation that adds a remarkable novel facet to the array of functions of the important biological effector NO^⋅.

https://doi.org/10.7554/eLife.43718.001

Humans use heat, cooling, and freezing to protect their foods from mold and bacteria. Many animals, including a wasp called the European beewolf, have also developed ways to store and preserve food. Female beewolves hunt honeybees. After paralyzing a bee, the beewolf takes the body into an underground chamber and lays an egg on it. When a larva hatches from the egg, it feeds on the bee. The warm, humid conditions in the chamber provide ideal conditions in which larvae can develop, but also encourage mold and bacteria to grow.

Previous research has uncovered two methods used by beewolves to fight off mold. In 2007, researchers discovered that the female beewolf coats her bee prey with a layer of fats. This prevents water loss and keeps the outside of the bee dry so that mold spores cannot grow. In 2010, a further study showed that the female beewolf grows helpful bacteria inside her antennae and transfers some to her young. The bacteria produce antibiotics that protect the larvae and their cocoons from mold. But these two strategies alone cannot explain the high survival rate of beewolf young. This suggests that the beewolves have at least one more strategy to prevent mold from growing.

Now, Strohm et al. – including some of the researchers involved in the 2007 and 2010 studies – show that beewolf eggs emit high levels of a gas called nitric oxide, which reacts with oxygen to form nitrogen dioxide. Nitrogen dioxide is part of the air pollution generated by cars and is harmful to many species in high concentrations. Nitric oxide also plays an important role for many biochemical processes in virtually all organisms, albeit in very low concentrations. The beewolf eggs produce comparatively huge amounts of this gas to fumigate their brood chambers and protect themselves and their food from mold.

Strom et al. then investigated how the eggs produce nitric oxide. The eggs appear to use the same enzyme that some other organisms use to produce nitric oxide. However, the wasp version of the enzyme contains a small modification that might explain why the eggs can produce the gas in such large amounts.

Learning more about how beewolves evolved different anti-mold strategies could help researchers to develop new antimicrobial treatments for medical applications. In addition, it is not yet clear how the wasp eggs survive in high concentrations of nitric oxide and nitric dioxide. Inflammation and some human diseases produce nitric oxide, killing nearby cells. Understanding how the beewolf eggs survive could therefore help to treat these cells or protect them from damage.

https://doi.org/10.7554/eLife.43718.002

Microbes pose a major threat to the health of all animals and plants. These have responded by evolving a great diversity of defenses including hygienic behaviors (Gilliam et al., 1983), antimicrobial chemicals (Herzner et al., 2013; Vilcinskas et al., 2013; Gross et al., 2008), complex immune systems (Hooper et al., 2012; Iwasaki and Medzhitov, 2010), and defensive symbioses (Kaltenpoth et al., 2005; Flórez et al., 2017). Besides such pathogenic effects, many bacteria and fungi are severe, but often neglected, competitors of animals for nutrients, thus prompting the evolution of mechanisms to preserve food sources (Janzen, 1977; Rozen et al., 2008).

Some animals are particularly prone to suffer from microbial attack due to (1) high abundance of potentially harmful microbes in their environment, (2) a microbe-friendly microclimate and/or (3) limited defense mechanisms. The progeny of many insect species develop under warm and humid conditions in the soil, where they are exposed to a high diversity of bacteria and fungi. Moreover, compared to adult insects, immature stages, in particular eggs, have usually reduced abilities to prevent microbial infestation due to, for example, a thin cuticle or an inability to groom (Wilson and Cotter, 2013; Tranter et al., 2014). The situation is even aggravated when eggs and larvae have to develop on limited amounts of provisions that are susceptible to attack by ubiquitous and fast growing putrefactive bacteria and mold fungi (Janzen, 1977; Arce et al., 2013).

Such hostile conditions prevail in nests of subsocial Hymenoptera like the European beewolf Philanthus triangulum (Hymenoptera, Crabronidae). The offspring of these solitary digger wasps develop in subterranean brood cells provisioned by the female wasps with paralyzed honeybee workers (Apis mellifera, Apidae, Hymenoptera) (Strohm and Linsenmair, 2000) (Figure 1A). The beewolf egg is laid on one of the bees, the larva hatches after three days, feeds on the bees for six to eight days, then spins a cocoon and either emerges the same summer or hibernates. The warm and humid microclimate in the brood cell promotes larval development but also favors fast growing, highly detrimental fungi (Engl et al., 2016). Without any countermeasures the provisions will be completely overgrown by mold fungi within three days (Figure 1B), and the beewolf larva becomes infested by fungi or starves to death (Strohm and Linsenmair, 2001; Herzner et al., 2011a).

We have previously documented two adaptations that beewolves have evolved to counter the detrimental effects of fungi on their brood. First, beewolf females reduce molding of the larval provisions by coating the paralyzed bees with ample amounts of unsaturated hydrocarbons (Herzner et al., 2007). This embalming changes the physicochemical properties of the preys’ surface causing reduced water condensation on the bees (Herzner and Strohm, 2007). Due to the deprivation of water, germination and growth of fungi is delayed by two to three days (Herzner et al., 2011b). Second, during the long period of overwintering in their cocoons beewolf larvae are protected by antibiotics on their cocoons (Kaltenpoth et al., 2005; Kroiss et al., 2010). Prior to oviposition, beewolf females apply a secretion containing symbiotic Streptomyces bacteria to the ceiling of the brood cell. The secretion is taken up by the larvae and incorporated into the silk threads of their cocoons. There, the bacteria produce several antibiotics that effectively protect the cocoon and, thus, the larvae against fungus infestation (Kroiss et al., 2010; Engl et al., 2018).

Despite the considerable effect of prey embalming, when removed from brood cells at least 50% of embalmed bees showed fungus infestation within six days after oviposition (Strohm and Linsenmair, 2001). Since in natural brood cells only around 5% of the progeny succumb to mold fungi (Strohm and Linsenmair, 2001), we searched for an additional antimicrobial defense mechanism that takes effect during the early stages of beewolf development.

Here we report on a unique antifungal strategy that is employed by beewolf eggs to defend themselves and their provisions against mold fungi. Employing bioassays we discovered that beewolf eggs emit a strong antifungal agent that we identified as the gaseous radical nitric oxide (NO^⋅). We characterize the amount, time course and temperature dependence of emission and show that synthetic NO^⋅ exerts a similar effect as the gas emitted by beewolf eggs. Furthermore, we tested whether there was an interaction of the gas emitted by the eggs and the embalming of the prey by beewolf females. Using histological methods, inhibition assays, and gene expression analysis, we elucidate a biosynthetic pathway for NO^⋅ synthesis in beewolf eggs. To explore the evolutionary background of this remarkable antimicrobial strategy, we sequenced the relevant gene and mRNA. Our findings reveal a novel function of the eminent and widespread biological effector NO^⋅ in providing an extended immune defense to the producer by sanitizing its developmental microenvironment.

Emission of an antifungal volatile by beewolf eggs

Thorough examination of beewolf nests in observation cages (Strohm and Linsenmair, 1994) revealed that within 24 hr after oviposition, a conspicuous pungent smell occurred that was clearly emanating from the eggs and disappeared by the time the larvae hatched. We hypothesized that this smell was due to an antifungal agent. When paralyzed honeybees from completed beewolf brood cells were incubated individually, bees carrying an egg showed significantly delayed fungus growth compared to bees without egg over the period from oviposition to cocoon spinning (Kaplan Meier survival analysis, Breslow test, day 0–11: Chi square = 12, df = 1, p=0.001; Figure 2A). This difference was also significant for the period from oviposition to the hatching of the larvae (day 0–3: Chi square = 9.5, df = 1, p=0.002), suggesting that this effect is not due to possible antifungal mechanisms of the larvae but that it is mediated by the egg. Considering the distinctive odor that emanated from the eggs, we tested whether the antifungal effect is caused by a volatile agent. Two experiments supported this assumption. First, provisioned bees without wasp eggs that were kept in artificial brood cells together with bees carrying an egg (but without physical contact) showed significantly delayed fungal growth compared to control bees that were kept alone (Breslow test, day 0–11: Chi square = 7.6 df=1, p=0.006; day 0–3: Chi square = 9.1, df = 1, p=0.003; Figure 2B). Second, when one of the most abundant mold species from infested beewolf brood cells, the fast growing Aspergillus flavus (Engl et al., 2016), was exposed to the volatiles presumably emanating from beewolf eggs on nutrient agar for three days, its growth was entirely inhibited, whereas it thrived in controls (for all observation times 24 hr, 48 hr, 72 hr: binomial test: N = 20, p<0.001, Figure 3). Notably, when the beewolf larvae were removed from the assays shortly after hatching (three days after oviposition), no fungal growth occurred in the exposed areas during another three days. A similar experiment showed that paralyzed honeybees alone did not show any antifungal activity (Appendix 1: Additional data 1). Analogous bioassays with five other fungal strains (A. flavus strain B, Mucor circinelloides, Penicillium roqueforti, Candida albicans and Trichophyton rubrum) revealed that in all cases fungus growth was likewise completely inhibited in the area that was exposed to volatiles from beewolf eggs, whereas the fungi thrived in the respective control areas (for each strain: N = 8, binomial test: p<0.01). We conclude that beewolf eggs release a volatile compound with broad spectrum fungicidal properties.

Identification of the antifungal volatile

The odor emanating from the eggs was similar to that of strong oxidants like chlorine, ozone and nitrogen dioxide (subjective evaluation of several observers, [Mücke and Lemmen, 2010]). In fact, the generation of a strong blue coloration when placing a beewolf egg into the lid of a reaction tube filled with a iodide/starch solution ([Jander and Blasius, 1971], see iodometry in Materials and methods) revealed the existence of an oxidant in the headspace of beewolf eggs. There are few gaseous oxidants that might be considered, in particular chlorine, ozone and nitrogen dioxide. Ozone has, to our knowledge, not been described to be synthesized by organisms. Molecular chlorine has been reported as an intermediate in some organisms but its occurrence seems to be restricted to phagocytosis (Hazen et al., 1996). The most likely candidate was the radical nitrogen dioxide (NO₂^⋅), because there is a plausible way for it being generated by wasp eggs: Insect embryos synthesize small amounts of nitric oxide (NO^⋅) as signaling effectors for developmental processes (Andersen et al., 2013). If such odorless NO^⋅ was emitted from the egg, it would spontaneously react with oxygen (Soegiarto et al., 2003; Mur et al., 2011) to yield the strong-smelling NO₂^⋅. Moreover, belonging to the reactive nitrogen species (RNS), NO^⋅ and NO₂^⋅ show considerable antimycotic activity (Fang, 1997; Lai et al., 2011) that would explain the observed fungicidal effect of beewolf eggs. Hence, we hypothesized that eggs synthesize and emit NO^⋅ that reacts with the oxygen in brood cells to NO₂^⋅ thus generating the pungent smell and the antimycotic activity.

We tested whether beewolf eggs produce and emit NO^⋅ and/or NO₂^⋅ by conducting a series of experiments. First, headspace samples of confined beewolf eggs were subjected to the Griess assay, the standard procedure for the specific detection of NO^⋅ and NO₂^⋅ (Tsikas, 2007). The emerging red color of the resulting azo dye (Figure 4—figure supplement 1) clearly indicated the presence of NO^⋅/NO₂^⋅. To visualize the emission of NO^⋅ from beewolf eggs, we sprayed a solution of an NO^⋅ specific fluorescent probe, Diaminorhodamin-4M AM (DAR4M-AM), onto prey bees carrying freshly laid eggs. The small droplets of the DAR4M-AM solution on the bees showed a clear fluorescence around the egg that increased over several hours (Figure 4B). No such effect was seen on control bees without eggs (Figure 4A). Moreover, beewolf eggs injected with the DAR4M-AM solution showed a strong fluorescence that peaked about one day after oviposition (N = 45, Figure 5A). The same treatment yielded only weak fluorescence in the eggs of two other Hymenoptera (the Emerald cockroach wasp, Ampulex compressa, N = 9, and the Red mason bee, Osmia bicornis, N = 12; Figure 5C and D) and in newly hatched beewolf larvae (N = 4, Figure 5—figure supplement 1). Autofluorescence of beewolf eggs injected with buffer only (N = 10) was negligible (Figure 5B). These findings strongly imply that beewolf eggs produce and release NO^⋅.

Amount and time course of NO^⋅ emission

Using iodometry, we determined that a beewolf egg (volume: 4.1 ± 0.5 mm³, N = 16) emits on average 0.25 ± 0.09 µmol NO^⋅ (N = 233). The rate of NO^⋅ production was initially very low, but increased to a distinct peak 14–15 hr (at 28°C) after oviposition (Figure 6); around 90% of NO^⋅ emission occurred within a two-hour period. Assuming no loss due to reactions or leaking out of the confined space of brood cells (volume 3.2 ± 0.9 cm³, N = 250), the nitrogen oxides would accumulate to average concentrations of 1690 ± 680 ppm. The timing of the onset of NO^⋅ emission was strongly temperature dependent (Figure 6—figure supplement 1), with higher temperatures resulting in an earlier NO^⋅production (temperature coefficient Q₁₀ = 2.74).

Antifungal effect of synthetic NO^⋅

To test whether the observed antifungal effect of the gas that is emitted by beewolf eggs was due to NO^⋅, we conducted an experiment using synthetic NO^⋅ but otherwise emulated natural conditions as closely as possible. Artificial brood cells containing a bee without egg were injected with either synthetic NO^⋅ to generate a peak concentration of 1500ppm or with nitrogen as controls. There was a significant delay in the onset of fungus growth on the bees exposed to synthetic NO^⋅ as compared to controls (Figure 7, Breslow test: Chi square = 13.3, df = 1, p<0.0001).

Combined effect of NO^⋅ emission and prey embalming

Since both, NO^⋅ emission and embalming of the prey bees by beewolf females take effect during the first days after oviposition, we assessed the antifungal effects of these defense mechanisms alone and in combination. Bioassays with bees in artificial brood cells that were either embalmed or not and carried an egg or not revealed that the onset of fungal growth differed significantly among treatment groups (Figure 8: Kaplan Meier survival analysis, Breslow test: Chi square = 69.6, df = 3, p<0.001). Pairwise comparisons showed that, on average, fungus growth was first detected on honeybees that had not been embalmed and did not carry an egg, second were prey items that were embalmed but did not carry and egg, third were not embalmed honeybees carrying an egg and least susceptible were embalmed honeybees with an egg (Breslow test for all pairwise comparisons: Chi square ≥8.6, df = 1, p≤0.003). The timing of conidia formation followed the same pattern (Breslow test: Chi square = 67.4, df = 3, p<0.001; for all pairwise comparisons: Chi square ≥4.5, df = 1, p≤0.034; Figure 8).

Synthesis of NO^⋅ in Beewolf eggs

Eukaryotes synthesize NO^⋅ from the amino acid L-arginine by the enzyme nitric oxide synthase (NOS) (Röszer, 2012) which is highly conserved also in insects (Regulski and Tully, 1995). The exceptional level of NO^⋅ emission of beewolf eggs raised the question of whether they employ the same pathway or have evolved a different mechanism. First, using histological staining, we assessed evidence for and site of NOS activity in beewolf eggs during the time of peak NO^⋅ emission. The fixation insensitive nicotinamide-adenine-dinucleotide phosphate (NADPH) -diaphorase assay resulted in strong blue staining only in embryonic tissue (Figure 9), thus indicating NOS activity. Second, by employing reverse transcription and real time quantitative PCR, we revealed that the temporal expression pattern of NOS-mRNA showed a clear peak around 19–20 hr after oviposition (Figure 10). For this experiment, the eggs were kept at 25°C, so the timing of peak NOS expression corresponds to the timing of peak NO^⋅ emission at this temperature (Figure 6—figure supplement 1). Third, to directly test for the involvement of NOS we injected beewolf eggs with Nω-nitro-L-arginine methylester (L-NAME), a NOS-inhibiting analog of L-arginine (Willmot et al., 2005). This treatment caused a significant decrease in NO^⋅ emission, whereas the non-inhibiting enantiomer D-NAME had no such effect (Figure 11).

The beewolf NOS-gene and NOS-mRNA in eggs

In contrast to vertebrates, most invertebrates appear to have only one type of NOS (Rivero, 2006; Whitten et al., 2007). Considering the high level of NO^⋅ production in beewolf eggs, we hypothesized that beewolves have more than one NOS gene or that the NOS responsible for the NO^⋅ synthesis in beewolf eggs might exhibit considerable changes in enzyme structure compared to the NOS of related species. Sequencing of the NOS-gene(s) of P. triangulum (Pt-NOS) revealed only one Pt-NOS copy in the beewolf genome comprising 9.36 kbp with 25 exons (Figure 11—figure supplement 1). A phylogenetic analysis of the resulting amino acid sequence revealed a high similarity to the NOS of the closely related bees (Apidae, Figure 11—figure supplement 2). However, mRNA sequencing showed that, in contrast to adult beewolves and honeybees, the NOS-mRNA of beewolf eggs (3.72 kbp) lacks exon 14 comprising 144 bp. In the NOS-mRNA of adult beewolves this exon is located between the binding domains for calmodulin and flavin mononucleotide (FMN) (Figure 11—figure supplement 1).

Fighting pathogens is of outstanding importance for any organism and has driven the evolution of a great diversity of antimicrobial defenses. Internal immune systems have been extensively documented especially in vertebrates (Akira et al., 2006; Hirano et al., 2011) but also in insects (Lemaitre and Hoffmann, 2007; Siva-Jothy et al., 2005), including insect eggs (Gorman et al., 2004). However, comparatively little is known about external antimicrobial strategies that provide protection for the own body, for the progeny, or for food. Mechanical grooming is an important mechanism to remove microbes (Zhukovskaya et al., 2013). There are some reports on the application of antimicrobial secretions on the body surface by adult insects (Wilson and Cotter, 2013; Otti et al., 2014) or inside a host by larvae of a parasitoid wasp (Herzner et al., 2013). Carrion beetles preserve the larval food, buried carcasses, by application of antimicrobials (Degenkolb et al., 2011) and by controlling the microbiome on the carcasses (Shukla et al., 2018). Females of some insect species deposit antimicrobial chemicals (Vander Meer and Morel, 1995; Marchini et al., 1997) or antibiotics producing symbiotic bacteria (Flórez et al., 2017; Flórez et al., 2015) onto their eggs and ant workers can counter microbial infestation of the brood by applying venom (Tragust et al., 2013). Recently, the employment of volatile antimicrobials by insects as a means of external defense has gathered some interest (Gross et al., 2008; Gross and Schmidtberg, 2009; Weiss et al., 2014; Lopes et al., 2015).

Like other insects that develop in the soil, beewolves are particularly menaced by a diverse and unpredictable range of detrimental microbes. In fact, beewolf progeny and their provisions are under severe threat from fast growing mold fungi (Strohm and Linsenmair, 2001). The development of beewolf progeny from oviposition to cocoon spinning lasts about 11 days and is, thus, rather fast. So even a few days delay in fungus growth provides a considerable benefit for the larvae. Beewolves have evolved at least three very different antimicrobial defenses that provide an effective, coordinated, and long-term protection against a broad spectrum of microbes during the whole development. First, throughout the long period of winter diapause prior to emergence progeny are protected by antibiotics on their cocoons that are produced by symbiotic Streptomyces bacteria (Kaltenpoth et al., 2005; Kroiss et al., 2010; Kaltenpoth et al., 2014). Second, during the early egg and larval stages, molding of the provisions is retarded by an embalming of the honeybees with lipids by the mother wasp (Strohm and Linsenmair, 2001). Third, as shown here, the emission of gaseous nitrogen oxide radicals by the beewolf egg results not only in delay of molding but in killing of detrimental fungi in their immediate environment thus, at least partly, eliminating this major threat.

The emission of a gaseous agent by beewolf eggs to their confined brood cells is an ideal way to sanitize such intricately structured surfaces as the bodies of honeybees and the rough walls of the brood cell. NO^⋅ seems to be a most suitable gaseous agent because it can obviously be produced by beewolf eggs in amounts that effectively kill mold fungi in their brood cell. Such volatile sanitation mechanisms that provide a front-line defense against microbes (Gross et al., 2008; Weiss et al., 2014; Lopes et al., 2015) will mostly be inconspicuous and might turn out to be a wider theme in nature.

Exact quantification of nitrogen oxides (NO^⋅ and NO₂^⋅) in beewolf brood cells on a micro scale or with time has not yet been accomplished. Brood cells are located in rather compact fine grained sandy soil with some moisture. Moreover, the walls of the nest burrows and the brood cells are covered with a layer of hydrocarbons (Kroiss et al., 2009) that might provide an additional barrier. Thus, brood cell walls are neither very porous nor are they sealed. Accordingly, the concentration of NO^⋅ and NO₂^⋅ in the brood cell will decrease but at a slow rate. By the time the larvae hatch (three days after oviposition) the smell of NO^⋅ has vanished, indicating that the nitrogen oxides have disappeared or at least decreased considerably, explaining why the larvae remain unaffected without actually being resistant to NO^⋅. However, even assuming some loss during the two hour period of peak NO^⋅ production the estimated maximum concentration of nitrogen oxides (NO^⋅ and NO₂^⋅) in beewolf brood cells (probably around 1500 ppm or 60 µmol/l) considerably exceeds the concentrations observed in animal tissues (mostly lower than 0.1 µmol/l [Wink et al., 2011], 0.85–1.3µmol/l in muscle tissue [Vaughn et al., 1998]). The maximum concentration in beewolf brood cells might be even higher than what is used in medical applications against multiple drug resistant bacteria (200 ppm NO^⋅[Ghaffari et al., 2006]) or in antifungal treatment of fruit (50–500 ppm NO^⋅[Lazar et al., 2008]) and is far beyond permissible exposure limits for humans (e.g. for the USA: 25 ppm for NO^⋅, 5 ppm for NO₂^⋅[Administration USOSaH, 2014]).

Synthetic NO^⋅ applied to artificial brood cells at a concentration of 1500ppm, the estimated concentration of nitrogen oxides in natural brood cells, significantly delayed fungus growth on bees. Since there was oxygen available in the brood cell, NO^⋅ was oxidized to NO₂^⋅ similarly to natural brood cells. The effect size (NO^⋅ treatment vs control, Figure 7: hazard ratio = 0.41, 95% confidence interval 0.198–0.845) was slightly lower than in a comparable experiment with eggs as the source of the antifungal gas (data for unembalmed bees from the experiment of a combined effect of embalming and emissions from the egg: Appendix 1—table 2: hazard ratio = 0.22, 95% confidence interval 0.1–0.47). However, since the confidence intervals of the hazard ratios are mutually overlapping, there is no evidence for a significant difference in the antifungal effect of 1500ppm synthetic NO^⋅ and the gas emitted by beewolf eggs. Although we cannot exclude that small amounts of other active volatiles are released by beewolf eggs, we thus conclude that the antifungal effect of brood cell fumigation by beewolf eggs is predominantly or exclusively due to NO^⋅ and its oxidation product NO₂^⋅.

Notably, the combination of prey embalming with unsaturated hydrocarbons that reduces condensation of water on the bees (Herzner and Strohm, 2007) and brood cell fumigation with NO^⋅/NO₂^⋅ seems to affect fungal growth beyond either of these antimicrobial measures alone. One possible explanation is based on the fact that NO^⋅ and NO₂^⋅ dissolve in water (confirmed by the spraying of a bee with a fluorescent dye, Figure 4) to yield nitric acid and nitrous acid, with the latter being a potent antibacterial agent (Gao et al., 2015). Although embalming reduces the amount of water condensation on the prey bees, some very small droplets occur. The concentration of nitrous acid in these droplets will be considerably higher than in the larger droplets that would occur without embalming. Thus, fungal germs that might have survived the NO^⋅/NO₂^⋅ atmosphere are not only impaired by limited availability of water, but the accessible water might be toxic for them. Moreover, due to the solubility of NO^⋅ and NO₂^⋅, abundant water droplets on the bees could reduce the concentration of these radicals in the brood cell, thus lessening their antimicrobial effect. The reduction of water on the bees due to prey embalming could thus help to keep fumigation effective. The combination of prey embalming and fumigation, thus, has a twofold effect. Many fungi will be killed by the NO^⋅/NO₂^⋅. The remaining spores will encounter unfavorable conditions that slow-down germination and growth so that the majority of larvae are able to consume most of their provisions without severe competition by mold fungi. Once larvae have spun their cocoon the antibiotics that are produced by the symbiotic bacteria take over protection until emergence (Kaltenpoth et al., 2005; Kroiss et al., 2010; Engl et al., 2018). Within this multifaceted antimicrobial strategy of beewolves, brood cell fumigation might be the most important component since it takes effect at a very early developmental stage and, thus, provides beewolf offspring with a decisive head start over the fast growing mold fungi.

NO^⋅ is an ancient biological effector of immense importance for all kinds of organisms ranging from prokaryotes to higher plants and animals (Röszer, 2012; Moroz and Kohn, 2007). Owing to its high diffusibility across biomembranes and specific chemical properties, this gaseous radical plays a crucial role in a multitude of biological processes (Röszer, 2012; Moroz and Kohn, 2007). In vertebrates, NO^⋅ is synthesized from L-arginine by three different isoforms of NOS that are encoded by different genes (Röszer, 2012; Moroz and Kohn, 2007). Low levels of NO (<1µmol/l) are produced by constitutive NOS (cNOS) isoforms (endothelial eNOS, neuronal nNOS) and have signaling functions, for example in neuronal development and in the regulation of vascular tone in vertebrates. Higher NO^⋅ concentrations (1–10 µmol/l, [Thomas et al., 2003]) are generated by an inducible NOS (iNOS). At such levels NO^⋅ is highly cytotoxic (Thomas et al., 2003), making it a powerful antimicrobial (Fang, 1997; Lai et al., 2011), for example in macrophages (Röszer, 2012). However, overproduction of NO^⋅ due to inflammatory processes (Filipović et al., 2010) or certain diseases (e.g. Alzheimer’s disease, [Lüth et al., 2002]) may cause harmful side-effects (Pacher et al., 2007) and even septic shock (Titheradge, 1999). Moreover, NO^⋅ might affect carcinogenesis and tumor progression in a positive as well as in a negative way (Burke et al., 2013).

In living tissues, NO^⋅ is usually removed within seconds by reacting with the heme group of molecules such as oxyhemoglobin (Beckman and Koppenol, 1996; Wink et al., 2011) (very low concentrations may still persist for hours [Moroz and Kohn, 2007]). In brood cells, there is enough oxygen (670 µl) to support the metabolism of the egg and of the paralyzed bee as well as the oxidation of NO^⋅ (for more details see Appendix 1: Additional discussion 1). In air, the autooxidation to NO₂^⋅ is comparatively slow so that NO^⋅ may persist (depending on its concentration) for several seconds to minutes (Mur et al., 2011; Wink et al., 2011) or even hours (Soegiarto et al., 2003). Thus, the NO^⋅ emitted by beewolf eggs might directly affect fungi, for exapmle by damaging DNA (Lai et al., 2011; Jones et al., 2010) or by reacting with the heme group of enzymes like cytochrome P450 and cytochrome c oxidase, thus inhibiting these crucial components of the mitochondrial respiratory chain (Thomas et al., 2003; Feelisch, 2008; Canessa and Larrondo, 2013). Yet, most of the antimicrobial activity of NO^⋅ is attributed to indirect effects via reactive nitrogen species (RNS), in particular nitrogen oxides (NO₂^⋅, N₂O₃) and peroxynitrite (ONOO^–, upon reaction with superoxide) (Thomas et al., 2003). NO₂^⋅, has been reported to be severely cytotoxic, for example by nitration of tyrosine residues and oxidation of proteins and lipids (Fang, 1997; Bogdan, 2001).

A beewolf egg of approximately 5 mg emits 0.25 µmol NO^⋅ within a period of about 2.5 hr, or 20.000 µmol/kg*h, a value that is about four orders of magnitude higher than reported baseline levels of NO^⋅ synthesis in humans (0.15 – ~ 4.5 µmol/kg*h [Castillo et al., 1996], rats (0.6–9 µmol/kg*h [Wu et al., 1999]) and plants (Arabidopsis thaliana, 0.36–3 µmol/kg*h [Zeidler et al., 2004]), and even considerably higher than in lipopolysaccharide (LPS)-activated macrophages (~800 µmol/kg*h, estimated from Wu et al., 1999). To investigate whether a NOS was involved in NO^⋅ production in beewolf eggs we conducted three experiments. First, a specific histochemical assay indicated NOS activity in embryonic tissue but not in other parts of the egg. Second, quantitative PCR revealed elevated expression of the NOS gene at the time of peak NO^⋅ production. Finally, competitive inhibition of NOS by L-NAME caused a significant reduction in NO^⋅ production. While each of the three results might have alternative explanations (e.g. L-NAME might not be a perfectly specific NOS inhibitor [Peterson et al., 1992]), taken together these findings provide strong evidence that a NOS, located in the embryonic tissue, is involved in NO^⋅ production of beewolf eggs.

Searching for possible adaptations that might accomplish this extremely high rate of NO^⋅ production the beewolf NOS gene was sequenced. Only one beewolf NOS (Pt-NOS) gene was found. The derived amino acid sequence did not reveal considerable differences compared to the NOS of the closely related bees (Figure 11—figure supplement 2). Thus, there is no evidence for extensive evolutionary changes with regard to the gene itself. Moreover, the structure of the Pt-NOS gene is largely homologous to other insects, for example Anopheles stephensi mosquitoes (Luckhart et al., 1998).

However, in contrast to adult beewolves, the NOS-mRNA in beewolf eggs lacks exon 14 (144 bp, Figure 11—figure supplement 1). Such alternative splicing that results in different NOS-mRNAs, including the deletion of exons (but others than in beewolves), has been documented in A. stephensi in response to Plasmodium infection (Luckhart and Li, 2001). Moreover, NOS splice variants may result in organ-specific enzymes in other organisms (Röszer, 2012). Presumably, beewolf eggs produce smaller amounts of another NOS splice variant to support signaling functions in the developing embryo.

In adult beewolves, the exon missing in the NOS-mRNA of eggs is located between the binding domains for calmodulin and FMN. Since calmodulin is believed to be responsible for NOS regulation (Smith et al., 2013) the deletion of an adjacent part might affect the control of NOS activity in beewolf eggs. Thus, the alternative splicing might enable the production of such large amounts of NO^⋅. Notably, compared to the cNOS (comprising eNOS, and nNOS) the inducible NOS isoform of vertebrates (iNOS) that generates higher concentrations of NO^⋅ to combat microbes lacks a section of about 40 amino acids (120 bp) near the FMN domain. Interestingly, this section is thought to be responsible for autoinhibition of the cNOS (Salerno et al., 1997) and its lack enhances NO^⋅ production by the iNOS. The conspicuous similarity between vertebrate iNOS and the NOS in beewolf eggs with regard to the length of the missing section and its position might suggest a convergent modification to achieve a NOS with high synthetic capacity. Whereas vertebrates have evolved another gene, beewolf eggs might accomplish a similar effect by alternative splicing of the mRNA.

The possible loss of regulation of the NOS and the pattern of Pt-NOS expression in the eggs suggest that in beewolf eggs the activity of the enzyme is regulated by gene expression like the NOS in Plasmodium infested A. stephensi (Luckhart et al., 1998) and the iNOS of vertebrates (Wong et al., 1996; Morris Jr, 1999). However, in contrast to these caes, in beewolf eggs expression of the Pt-NOS seems not to be induced by immunostimulants but to occur obligatorily at a certain stage in the development of the beewolf embryo. While we cannot exclude that there is an additional, yet unknown, pathway of NO^⋅ production in beewolf eggs, we hypothesize that the NOS and in particular its alternative splice variant plays a significant role in brood cell fumigation by beewolf eggs.

However, even the combined effect of prey embalming and brood cell fumigation does not provide perfect protection as fungus infestation still causes larval mortality in 5% of the brood cells in the field (Strohm and Linsenmair, 2001). Some fungal spores might survive under the bees because they were screened against the gas. Another possibility, namely that strains of the ubiquitous mold fungi that are the main causes of molding in beewolf brood cells (Engl et al., 2016), have evolved resistance against the toxic effects of NO^⋅/NO₂^⋅ seems rather unlikely. Ultimately, there will be only weak selection for resistance at all since beewolf brood cells are certainly a rare habitat for the ubiquitous mold fungi (Engl et al., 2018). Moreover, there will be no repeated exposure of the same fungal strains to fumigation that would be required to favor the evolution of resistance. While there are examples for detoxification of lower concentrations of NO^⋅ (mainly by scavengers like flavohemoglobins) in different fungi, including species of Aspergillus (Martins et al., 2011; Zhou et al., 2009), the NO^⋅/NO₂^⋅ levels emitted by beewolf eggs are very high and likely affect several very basic biochemical processes, thus making the evolution of an effective resistance unlikely.

While brood cell fumigation clearly retards molding of larval provisions, the antimicrobial effect of NO^⋅ and NO₂^⋅ might harm the symbiotic Streptomyces bacteria that beewolf females apply to the brood cell prior to egg laying (Kaltenpoth et al., 2005; Kroiss et al., 2010). Since the symbiotic bacteria are important for the survival of larvae in the cocoon and are vertically transmitted from beewolf mothers to their daughters (Kaltenpoth et al., 2014), a considerable number of symbionts have to survive the brood cell fumigation. At the moment we can only speculate how the bacteria can survive. Conceivably, because of strong selection due to specialization and repeated exposition, the symbiotic bacteria have evolved mechanisms to cope with the high concentrations of NO^⋅/NO₂^⋅ (Poole, 2005; Wareham et al., 2018). Possibly, the fumigation slowly evolved after the establishment of the symbiosis; thus bacteria might have been able to gradually evolve resistance. Moreover, the bacteria are applied to the ceiling of the brood cell, which might reduce negative effects of the nitrogen oxides since these are heavier than air (Lide, 1995) and will accumulate in the lower part of the brood cell. Additionally, the bacteria are embedded in copious amounts of a secretion consisting of mostly unsaturated hydrocarbons (Kaltenpoth et al., 2009) that might shield the bacteria from the fumigants. Finally, host- and/or symbiont derived antioxidants in the hydrocarbon matrix could detoxify NO^⋅ and NO₂^⋅ and protect the symbiotic Streptomyces bacteria.

How could brood cell fumigation with high concentrations of NO^⋅/NO₂^⋅ have evolved? Generally, it has been assumed that the primary purpose of NO^⋅ was signaling at low concentrations and that the antimicrobial functions of higher concentrations are derived (Fang, 2004). Assuming a similar scenario for beewolves, small amounts of NO^⋅ that were originally produced for developmental processes (Andersen et al., 2013) might have accidentally been released into the confines of the subterranean brood cell and slightly affected the germination or growth of fungi by interfering with regulatory processes (Röszer, 2012; Wang and Higgins, 2005). Given the severe threat posed by microbes, such initial benefits would have caused strong selection for elevated NO^⋅ emission by the eggs. This would have considerably increased progeny survival and might have allowed ancestral beewolves to nest in an expanded range of habitat types, including nesting sites with high risk of microbial infestation, or to exploit highly susceptible but readily available prey species. Brood cell fumigation with large doses of NO^⋅ thus represents a key evolutionary innovation. Since NO^⋅ is used as an antimicrobial in the immune systems of many animals (Bogdan et al., 2000), its deployment as an antifungal gas can be viewed as an innate, externalized immune defense of beewolf eggs. Such externalized components of the immune system have recently been recognized as important and possibly widespread antimicrobial measures (Otti et al., 2014).

The clear benefit of brood cell fumigation, however, is probably accompanied by substantial costs in terms of energy and biochemical resources (Rivero, 2006). NO^⋅ is synthesized from L-arginine, an amino acid that is an important constituent of many proteins and biochemical pathways (Morris Jr, 2000) and it is an essential amino acid for most insects (Barbehenn et al., 1999; Payne and Loomis, 2006) (e.g. phytophagous insects [Berenbaum, 1995], mosquitos [Uchida, 1993], aphids [Sasaki and Ishikawa, 1995; Akman Gündüz and Douglas, 2009], butterflies [Erhardt and Rusterholz, 1998; O’Brien et al., 2003], true bugs [Mesquita et al., 2015], parasitoid wasps [Thompson, 1976; Barrett and Schmidt, 1991], bees [de Groot, 1952; Weiner et al., 2010]). Thus, beewolves have either evolved the capacity to synthesize L-arginine or female beewolves have to provide each egg with sufficient L-arginine for both brood cell fumigation and embryogenesis. Moreover, NO^⋅ synthesis by NOS requires the cofactors flavin adenine dinucleotide (FAD), FMN, (6R-)5,6,7,8-tetrahydrobiopterin (BH4) and NADPH (Förstermann and Sessa, 2012), thus competing with other metabolic pathways in the developing beewolf embryo.

One of the most remarkable aspects of our study is that the embryos inside the egg survive the high concentrations of toxic nitrogen oxides during synthesis and emission as well as after its release to the brood cell. This is all the more surprising since beewolf larvae that were accidentally exposed to the gas emitted by eggs died (Strohm, unpublished observations). The synthesis and emission of such high amounts of NO^⋅ likely requires a number of concomitant adaptations that protect beewolf embryos against the cytotoxic effects of high concentrations of NO^⋅ and NO₂^⋅. One possibility is the employment of carrier molecules to transfer NO^⋅ to the egg shell. In blood sucking hemipterans, for example, nitrophorins carry NO^⋅ to its release site to dilate blood vessels (Davies, 2000). The mechanistic basis of NO^⋅ tolerance of beewolf eggs is of particular interest, since excessive production of NO^⋅ due to inflammatory processes (Guzik et al., 2003) or certain diseases (e.g. Alzheimer’s disease, [Lüth et al., 2002; Pacher et al., 2007; Calabrese et al., 2007; Pautz et al., 2010]) might cause severe pathological complications in humans. Thus, understanding how beewolf eggs avoid the toxic effects of NO^⋅ might inspire the development of novel medical applications.

Our findings reveal a surprising adaptation in a mass-provisioning digger wasp to cope with the threat of pathogen infestation in the vulnerable egg and larval stages. Sanitizing the brood cell environment by producing high amounts of NO^⋅ significantly enhances the survival of immatures by reducing fungal growth on their provisions. Given that mass-provisioning and development underground are widespread ecological features among digger wasps and bees and considering the difficulties of detecting volatiles in subterranean nests, such gaseous defenses might be more widespread and as yet underappreciated. In addition to revealing new perspectives on antimicrobial strategies in nature and amplifying the biological significance of NO^⋅, beewolves offer unique opportunities to elucidate general questions on the evolution and regulation of NOS as well as the production of and resistance to high concentrations of NO^⋅.

Animals

Beewolf females, Philanthus triangulum F. (Apoidea, Crabronidae), were either caught in the field from populations in Franconia (Germany) or were the F1 progeny of such females kept in the laboratory. They were housed in observation cages (Strohm and Linsenmair, 1994) that provided access to newly completed brood cells. The cages were placed in a room with temperature control (20–22° at night, 25–28°C in the daytime) and were lit for 14 hr per day by neon lamps. Honeybees, Apis mellifera L. (Apoidea, Apidae), the females’ prey, were caught from hive entrances or from flowers and provided ad libitum. Honey was provided ad libitum in the flight cage for the nutrition of both honeybees and beewolf females.

To obtain freshly laid eggs, observation cages were checked hourly. Completed brood cells were opened, their length and width was measured using calipers and the egg and/or honeybees were removed and used for the experiments. Brood cell volume was estimated as a prolate spheroid with brood cell length as the major and width as the minor axis. The bees in brood cells had been paralyzed, embalmed with lipids (Herzner et al., 2007), and provisioned by beewolf females. Egg volume was estimated by calculating the volume of a cylinder with the respective length and width of an egg (both determined using a stereomicroscope with eyepiece micrometer). The temperatures at which eggs were kept reflect natural conditions (Herzner and Strohm, 2008) and allow for an optimal development (Strohm, 2000).

General experimental procedures

For all experiments beewolf eggs were harvested from brood cells of various females. Eggs were randomly allocated to different treatment groups. Sample sizes refer to independent biological replicates, that is each replicate represents a different egg or brood cell – with the exception of quantitative PCR, where several eggs were pooled for one sample (see below). As it is very demanding to obtain beewolf eggs, the availability of eggs of a certain developmental stage was limited. Generally, we used as many eggs as feasible (e.g. for quantitative PCR). For some experiments we decided on a meaningful sample size based on experience from preliminary experiments (e.g. we already knew that inhibition assays with beewolf eggs in Petri dishes were really clear-cut and required only few replicates). Moreover, due to the limited availability of beewolf eggs on a given day, replicates were conducted consecutively over several days.

Fungus inhibition assays

To test whether the time course of fungus growth on bees differed between those carrying an egg and those without egg, we used brood cells (N = 22) that had been provisioned with two bees. We placed each bee individually into an artificial brood cell of natural shape and volume in sand-filled Petri dishes (diameter 10 cm) and with moisture levels similar to natural conditions. Petri dishes were placed in a climate chamber at 25°C in the dark. Bees were carefully checked visually every 24 hr for fungus growth without opening the Petri dishes. First signs of fungus infestation (hyphae) were recorded. The experiment was terminated after eleven days since all larvae had finished feeding and spun a cocoon by then. Since these are time event data, we used survival analysis (Kaplan Meier, Breslowe test; hazard ratios and their 95% confidence intervals are presented as estimates of effect sizes, SPSS Statistics 24) to compare the timing of fungus infestation of the bees with and without an egg. Larvae hatched on the third day after oviposition and started to feed on the bee. There was no evidence that hatched larvae were able to prevent fungus growth on the bee they occupied or others in the brood cell. However, to take a possible effect of the larva on the experimental bee into account, we carried out the analysis not only over the whole period from oviposition until the larvae spun into a cocoon (11 days) but also for the period from oviposition to the hatching of larvae (3 days). A significant difference already until the third day indicates that this effect was associated with the egg.

We examined whether beewolf eggs emit a volatile antimicrobial by conducting two experiments. For the first test, we used brood cells (N = 16) that contained three bees. The bees were transferred to artificial brood cells in sand-filled Petri dishes as described above. The bee with the egg and one of the bees without egg (the experimental bee) were placed together in the same artificial brood cell but without physical contact. The other bee without egg (the control) was kept alone in another artificial brood cell (in another Petri dish). We monitored the timing of fungus infestation as described above. We used survival analysis as described above. Again, to take an (unlikely) effect of the larva into account, we also carried out the analysis for the period from oviposition until the larvae hatched (day 3 after oviposition). A significant difference already until day three could only be caused by volatiles emanating from the egg.

For the second assay, we exposed conidiospores of a diverse spectrum of fungi to the volatiles emanating from beewolf eggs. Petri dishes (10 cm) containing culture medium (malt extract agar or Sabouraud-agar [Atlas, 2004]) were inoculated with conidia from different fungal strains (Aspergillus flavus strain A, Trichocomaceae, that was isolated from infested beewolf brood cells, [Engl et al., 2016], N = 20; A. flavus strain B, Mucor circinelloides, Mucoraceae; Penicillium roquefortii, Trichocomaceae; Candida albicans, Saccharomycetaceae; Trichophyton rubrum, Arthrodermataceae; N = 8 for all the latter strains and species; these were kindly provided by the Department of Hygiene and Microbiology of the Würzburg University Hospital). Conidiospores were harvested by sampling mature fungus colonies that were reared from stock cultures. A suspension of the conidia in sterile water was evenly distributed on the Petri dishes to obtain uniform growth of fungi. To recreate the concentrations of potential antibiotic volatiles in the brood cell, we used small plastic caps (3 ml, about the size of a brood cell) to confine test areas on the agar. Freshly laid eggs were placed singly on the bottom of a cap where they readily attached due to their natural stickiness. Each cap was then placed on a freshly inoculated Petri dish so that the agar under the cap was not in contact with the egg but was exposed to volatiles that emanated from the egg. An empty cap was placed on the same Petri dish as a control. The Petri dishes were incubated in a dark climate chamber at 25°C. Fungus growth under the experimental and control caps was recorded after 24, 48 and 72 hr. After 72 hr the caps with the hatched larvae were removed, and fungal growth was further recorded after another 24, 48 and 72 hr. Since the results were clear-cut with either no fungal growth or substantial growth (Figure 3) and no intermediate cases, the experimental and control areas were compared using binomial tests (software PAST [Hammer et al., 2001]).

Identification of the antimicrobial volatile

We hypothesized that nitric oxide (NO^⋅) and its main reaction product with oxygen, nitrogen dioxide (NO₂^⋅), were the most likely compounds emanating from beewolf eggs. The standard test for the detection of NO^⋅ and NO₂^⋅ employs the Griess reaction. We used a solution of sulfanilic acid and N-(1-naphthyl)-ethylenediamine (Spectroquant Nitrite Test, Merck, Germany, according to the manufacturer’s instructions). The Griess reagent specifically reacts with the nitrite anion (NO₂^–) to form a distinctive red azo dye (Guevara et al., 1998). NO^⋅ reacts with water to form nitrous acid (HNO₂) and can thus be directly verified by the Griess reaction. NO₂^⋅, however, disproportionates in water into nitrous acid and nitric acid (HNO₃) and the latter must be reduced to nitrous acid to react with the Griess reagent. Freshly laid beewolf eggs (collected within 2 hr after oviposition, N = 11) were placed in the lid of a 1.5 ml reaction tube where they readily attached due to their natural stickiness. Tubes without eggs (N = 11) were used as controls. Then 1 mL of the Griess test solution was added to the tube. For another sample (N = 15) the nitrate, which might be present in the solution, was reduced to nitrite by placing a glass fiber filter disc with small amounts of zinc powder (Jander and Blasius, 1971) on the surface of the solution. The same setting without an egg was used as control (N = 15). The tubes were incubated at 25°C for 24 hr, and the occurrence of the red coloration was examined visually and with a photometer (at 520 nm, Nanophotometer, Implen, Germany, quantitative measurements were not meaningful with this set-up since the azo dye is not perfectly stable over time, according to the manufacturer’s instructions). The samples with and without nitrate reduction showed qualitatively the same results.

NO^⋅ can also be detected by specific fluorescent probes. In particular, diaminorhodamin-4M AM (DAR4M-AM), a cell permeable, photostable fluorescent dye, has a high sensitivity and specificity for NO^⋅[Kojima et al., 2000]). A DAR4M-AM (Alexis Biochemicals, USA) solution was prepared according to the supplier’s instructions (10µmol/l in 0.1 mol/l phosphate buffer, pH 7.4). To verify and to visualize the emission of NO^⋅ from the egg, paralyzed honeybees either with freshly laid eggs (N = 8) or controls without eggs (N = 8) were sprayed with the DAR4M-AM solution using a nebulizer (the egg itself was screened from droplets during spraying) and kept in the dark (at 25°C in artificial brood cells as described above). After 20 hr, the bees were examined under a fluorescence microscope (Axiophot II, Zeiss, Germany, filter set 43: excitation 520–570 nm, emission 535–675 nm) and digital photos were taken (Nikon DS-2 Mv, Nikon Japan) at constant exposure times, to allow comparison of fluorescence intensity. Due to the size of the bees, several pictures had to be taken in the X,Y plane as well as along the Z axis. Pictures along the z-axis were stacked using the software Combine-ZP (www.hadleyweb.pwp.blueyonder.co.uk). Then these stacks were stitched using Photoshop Elements 5 (PSE5, Adobe Systems Inc USA). Since small peripheral background parts within the frame of the stacked and stitched picture were ‘empty’ these parts were filled with other background parts by using the clone stamp tool. Images were corrected for contrast and sharpness using PSE5 with identical settings for experimental and control specimens.

DAR4M-AM can also be used to detect NO^⋅ in tissues. Aliquots of 0.1–0.5 µl of the DAR4M-AM solution (see above) were injected into beewolf eggs (within 1 hr after oviposition, N = 64, in N = 45 eggs the embryo survived and developed) with a custom made microinjector equipped with glass capillaries (Eppendorf Femtotips II, Eppendorf, Germany) under microscopic control. Control eggs injected with buffer only (N = 10) were monitored in the same way to assess autofluorescence. For comparison, eggs of two other Hymenoptera (Osmia bicornis, Apoidea, Megachilidae, N = 12, and Ampulex compressa, Apoidea, Ampulicidae, N = 9; eggs from both species were obtained from our own laboratory populations) as well as freshly hatched beewolf larvae (N = 4) were injected with the DAR4M-AM solution. All eggs were kept in dark chambers at 25°C (a temperature within the optimal range for development for all these species), and fluorescence was observed directly after injection and 1, 3, 5, 24, sometimes 48 and 72 hr later. For some eggs not all time points were available. Fluorescence was examined under a fluorescence microscope and documented with a digital camera as described above. Contrast and sharpness of the images were optimized using Photoshop Elements 5 (Adobe, USA) with identical settings for all specimens.

Iodometry: Quantification, time course and temperature dependence of NO^⋅ production

Iodometry provides a simple but sensitive, reliable and precise method to quantify strong oxidants. To assess the amount of emitted nitrogen oxides, we placed freshly laid eggs (N = 233) individually into the lid of 1.5 ml reaction tubes where they readily attached due to their natural stickiness. Then 1 ml of a potassium iodide-starch solution (containing 1% KI and 1% soluble starch in distilled water) was added, the reaction tube was closed and kept for 24 hr at 28°C in a dark climate chamber. Oxidation of iodide results in iodine that forms a blue complex with starch (Jander and Blasius, 1971). The degree of coloration was quantified by measuring the absorbance at 590 nm in a spectrophotometer (Uvikon 860, Kontron, Germany). To assess the absolute amount of the oxidant, the solutions were subsequently calibrated by titration with a reference solution of sodium thiosulfate (concentration: 0.001 M; Merck, Germany) until the blue color of the iodine-starch complex disappeared.

To establish the time course of gas production, individual beewolf eggs (N = 4) were transferred within 1 hr after oviposition into the lid of reaction tubes and kept in a dark climate chamber at 28°C. Every hour, the cap with the egg was transferred to another reaction tube with fresh iodide-starch solution. Immediately after removal of the egg from a reaction tube, absorbance of the solution was measured at 590 nm as described above.

To investigate the temperature dependence of gas production, tubes with a newly laid egg and iodide-starch solution (as described above, N = 33 in total) were placed in a rack (with white background) inside a climate chamber and incubated at seven different constant temperatures (20, 22.5, 24, 25.5, 27, 28.5°C and 30°C). The time course of coloration of the iodide-starch solution was recorded using a digital camera (Canon EOS 20D, Canon, Japan) programmed to take pictures at 30 min intervals. The onset of gas production could be easily determined since the color of the solution turned from clear to dark blue from one picture to the next, that is within a 30 min interval. A quadratic regression curve was fitted to the data (SPSS Statistics 24) and the Q₁₀ value for the temperature dependence was estimated.

Bioassay to test for the antifungal effect of synthetic NO^⋅

We assessed the effect of synthetic NO^⋅ on the beginning of fungus growth on honeybees in artificial brood cells. Sand filled Petri dishes with artificial brood cells (volume 3 ml) were prepared as described above and a honeybee (collected at the entrance of a bee hive and killed by freezing) was placed into the brood cell. Nitric oxide was generated by the oxidation of zinc powder with nitric acid (HNO₃) (Jander and Blasius, 1971). In order not to affect the composition of the gases in the artificial brood cell, we adjusted the concentration of the generated NO^⋅ so that an addition of 10% of the volume of the brood cell resulted in an initial concentration of 1500ppm NO^⋅. Employing iodometry as described above, we adjusted the amounts of reactants so that the addition of 300 µl to the brood cell volume of 3 ml resulted in a NO^⋅ concentration of 1500ppm (the presumable peak concentration in natural brood cells). Zinc powder (0.5 g) was placed in a vial (20 ml) and the vial was closed with a plastic lid with two small holes (~0.5 mm, one for pressure compensation). Then the vial was extensively flushed with pure nitrogen to remove oxygen that would otherwise oxidize NO^⋅ to NO₂^⋅. Immediately, 150 μL of 20% HNO₃ were added with an insulin syringe so that the resulting gas mixture in the vial was composed of 15000ppm NO^⋅ and 98.5% N₂. Using a gastight syringe (Hamilton, Reno, NV, USA) an aliquot of 300 µl of this gas mixture was injected into the artificial brood cell with a bee through a small hole (~0.5 mm) in the lid of the Petri dish and the hole was immediately closed with adhesive tape (N = 20). Thus the concentration of NO^⋅ in the artificial brood cell was 1500ppm. As controls, otherwise identically prepared Petri dishes with bees in artificial brood cells were injected with 300 µl of pure nitrogen (N = 20).

The Petri dishes were incubated in a dark climate chamber at 25°C. All bees were carefully checked for fungus growth under a stereomicroscope for three days (twice per day). As a consequence, first signs of fungus growth were detected earlier than in other experiments of this study, where we used the unaided eye. Data were analysed using survival analysis as described above. To assess whether synthetic NO^⋅ has a similar antifungal effect as the gas emitted by beewolf eggs we compared the effect size (hazard ratio) of this experiment with the data testing for the effect of the egg produced gas on unembalmed bees (as part of the experiment on the ‘combined effects’, see below). If the 95% confidence intervals of the hazard ratios overlap, there is no evidence for a difference between the effects.

Bioassay to test for a combined effect of NO^⋅ and embalming

Fungus growth and conidia formation on bees of four different experimental groups were recorded for eleven days (until the larvae had spun their cocoons). The groups consisted of: (1) paralyzed honeybees that were not embalmed and did not carry an egg (n = 25), (2) paralyzed and embalmed honeybees without egg (n = 68), (3) paralyzed honeybees that were not embalmed but an egg was carefully transferred onto them from another bee (n = 21), and (4) paralyzed and embalmed honeybees with egg (n = 21). To control for effects of the transfer of eggs in group (3), each egg in group (4) was sham treated by using tweezers to lift it up from the bee and putting them back onto the same bee. Non-embalmed bees were removed from beewolf females immediately after paralysation. Embalmed bees were removed from brood cells in observation cages within 12 hr after oviposition. All bees were transferred to artificial brood cells (one bee per brood cell) in Petri dishes filled with moist sand. The Petri dishes were incubated in a dark climate chamber at 25°C. All bees were checked daily for both fungus growth and formation of conidia under a stereomicroscope. As above, first signs of fungus growth were detected earlier than in the experiments of this study, where we used the unaided eye. Data were analysed using survival analysis as described above with pairwise comparisons of treatment groups (SPSS 24).

Detection of NOS activity in egg tissue

To assess whether there was NOS activity in the egg tissue and where it was located, we used fixation-insensitive NADPH diaphorase staining with nitroblue tetrazolium (Virgili et al., 2001; Müller, 1994). Eggs were fixed in PBS containing 4% paraformaldehyde for 2 hr at 4°C, followed by cryoprotection in PBS with 12% sucrose for 20 hr. The tissue was soaked in Tissue Tec (Sakura Finetek, Netherlands) for 30 min, frozen, and 10 µm sections were cut on a cryostat microtome (CM3000, Leica, Germany). The sections were incubated for 60 min at 30°C with 50 mmol/l Tris-HCI, pH 7.8, 0.1% Triton X-100, and 0.2 mmol/l nitroblue tetrazolium chloride in the presence or absence (each N = 5) of 0.2 mmol/l β-NADPH to demonstrate fixation-insensitive NADPH diaphorase activity. The sections were dehydrated, mounted with Depex (Serva, Germany) and observed under a compound microscope (Zeiss Axiophot II). Photos were taken with a digital camera (Nikon DS-2 Mv). Since the egg was larger than the field of view of the camera, two pictures had to be taken and were stitched (Photoshop Elements 5, Adobe USA). Contrast and sharpness were optimized.

Phenology of NOS gene expression

If NOS is responsible for NO^⋅ production in beewolf eggs, the time pattern of NOS gene expression should largely resemble the time course of NO^⋅ production by showing a pronounced peak several hours after egg laying (the timing of the peak depending on temperature). We used reverse transcription and real time quantitative PCR to quantify the NOS mRNA in beewolf eggs at different times after oviposition. Since the amount of mRNA that could be obtained from single eggs was insufficient to get reproducible results, we conducted two trials for each of four different time intervals after oviposition (4–5, 9–10, 14–15 and 19–20 hr after oviposition). For each trial and per each time interval we pooled 25 eggs (all kept at 25°C), as well as 25 freshly hatched larvae repsectively. The eggs and larvae were removed from the brood cells at the specified times, shock frozen with liquid nitrogen and stored at −80°C. The RNA of each sample was extracted using the peqGOLD total RNA Kit (Peqlab, Germany) according to the supplier’s instructions and eluted with 20 µL RNase free water. An aliquot of 3 µL of the RNA was digested with DNaseI (Fermentas, Lithuania) and transcribed into cDNA with BioScript (Bioline, Germany) using an Oligo-dT primer (Fermentas, Lithuania) in a final volume of 20 µL. As a reference for basic levels of gene expression during the experimental period, mRNA of the housekeeping gene β-actin was quantified and the ratio of NOS/β-actin mRNA was calculated for each sample.

For quantitative PCR, we established new primers for both the NOS and β-actin genes of P. triangulum (based on the complete NOS sequences, see below) (NOS_qPCR_F1 and R4; Actin_qPCR_F1 and R1, Supplementary file 1). All primers were intron-overlapping to avoid the measurement of contaminating genomic DNA. The NOS and actin primers amplified fragments of 312 bp and 321 bp, respectively. The specificity of both primer sets was confirmed by sequencing purified PCR products. The qPCRs were performed on an Eppendorf Realplex cycler (Eppendorf, Germany) in a final volume of 25 µL, containing 1 µL of template cDNA (1 µL of the 20 µL RT reaction mix), 2.5 µL of each primer (10 pmol/l) and 12.5 µL of SYBR Green Mix (SensiMixPlus SYBR Mit, Quantace, UK). Standard curves were established by using 10⁻⁹ – 10⁻³ ng of PCR products as template. A NanoDrop TM1000 spectrophotometer (Peqlab, Germany) was used to measure DNA concentrations of the templates for the standard curves. PCR conditions were as follows: 95°C for 5 min, followed by 50 cycles of 56°C (β-actin) or 65°C (NOS) for 60 s, 72°C for 60 s and 95°C for 60 s. Then a melting curve analysis was performed by increasing the temperature from 60°C to 95°C within 20 min. Based on the standard curves, the amount of NOS and β-actin template and their ratio was calculated.

NOS inhibition assay

To verify the role of NOS in NO^⋅ production by beewolf eggs, we used an inhibition assay (Willmot et al., 2005). Since L-arginine is the substrate for NO^⋅ production by NOS, we injected either an inhibiting L-arginine analog or, for controls, a non-inhibiting enantiomer into freshly laid beewolf eggs. Chemicals were dissolved in 0.1 mol/l phosphate buffer pH 7.4. Using a microinjector (see above) eggs were injected with about 0.2 µl of 1.5 mol/l solutions of (1) the competitive inhibitor Nω-nitro-L-arginine methylester (L-NAME, Sigma-Aldrich, USA) (experimental group, N = 14), or (2) the non-inhibiting Nω-nitro-D-arginine methylester (D-NAME, Sigma-Aldrich, USA) (control group 1, N = 9) or (3) not injected at all (N = 14, control group 2). Each egg of the three groups was placed individually in the lid of a reaction tube with an iodide-starch solution as described above and incubated for 24 hr at 28°C. Then NO^⋅ production was assessed by measuring absorbance of the solution with a photometer (Implen Nanophotometer) at 590 nm. Statistical comparison of the groups was conducted using Mann-Whitney U-tests with correction after Holm (Holm, 1979) (SPSS Statistics 24).

Sequencing of the P. triangulum nitric oxide synthase gene (Pt-NOS) and mRNA

DNA was extracted from female beewolf heads with the Epicentre MasterPure Complete DNA and RNA Purification kit (Epicentre, USA) according to the manufacturer’s guidelines for tissue extraction. Eggs for RNA extraction were kept at a temperature of 27.5°C (range 26–29°C), collected 14–15 hr after oviposition, immediately frozen in liquid nitrogen and stored at −70°C until RNA extraction. Twenty eggs were pooled for extraction and homogenized by repeatedly pipetting in lysis buffer of the PeqGOLD Total RNA kit (Peqlab, Germany). Samples were processed according to the kit manual and frozen at −70°C. For the full transcriptome sequencing (to obtain the 5’ terminal region) RNA was extracted from the antennae of eight frozen female beewolves according to manufacturer’s protocol 1 of the innuPrep RNA Mini Kit (Analytik Jena, Germany).

Most of the beewolf NOS gene was amplified and sequenced by primer walking. Sequencing reactions were performed by a commercial service (Seqlab, Germany). Four degenerate primers (NOS860fwd2, NOS1571rev1, NOS_seq_F1_deg, and NOS_seq_R1_deg) were designed (Supplementary file 1) based on published NOS sequences of Drosophila melanogaster (U25117.1), Apis mellifera (AB204558.1), Anopheles stephensi (AH007775.1), Rhodnius prolixus (U59389.1), Manduca sexta (AF062749.1) and Nasonia vitripennis (NM_001168232.1). First, the central region (~700 bp, between NOS860fwd2 and NOS1571rev1) was amplified and sequenced. Based on this sequence, we designed a pair of P. triangulum specific primers (NOS_qPCR_F2 and NOS_qPCR_R2, Supplementary file 1). Using one specific central and one degenerate terminal primer (NOS_seq_F1_deg and NOS_seq_R1_deg, Supplementary file 1), respectively, fragments of 4–5 kb were amplified and sequenced by primer walking, which yielded the central 9.5 kb of the NOS gene.

Fragments larger than 2 kb were amplified with the PeqGOLD Mid-Range PCR System on a thermocycler (TGradient, Biometra, Germany). Reaction volumes of 12.5 µL contained 1 µL DNA template, 50 mmol/l Tris-HCl (pH 9.1), 14 mmol/l (NH₄)₂SO₄, 1.75 mmol/l MgCl₂, 350 mmol/l of each dNTP, 400 mmol/l of each primer and 0.5 U ‘MidRange PCR’ enzyme mix. An initial 3 min melting step at 94°C was followed by 35 cycles of 0.5 min at 94°C, 0.5 min at 58°C and 3 min +20 s per cycle at 68°C and a final extension time of 20 min at 68°C.

Fragments up to 2 kb were amplified using the PeqGOLD Taq. Reaction volumes of 12.5 µL contained 1 µL of DNA template, 50 mmol/l Tris-HCl pH 9.1, 14 mmol/l (NH₄)₂SO₄, 3 mmol/l MgCl₂, 240 µmol of each dNTP, 800 nmol/l of each primer and 0.5 U Taq. An initial 3 min melting step at 95°C was followed by 35 cycles of 1 min at 95°C, 1 min at 60°C and 2 min at 72°C and a final extension time of 3 min at 72°C.

The 3’ terminus was sequenced following the 3’ RACE protocol (Sambrook and Russell, 2001a). Briefly, cDNA was generated by reverse transcription with a poly-T primer. Before reverse transcription, co-extracted DNA was digested using DNaseI (New England Biolabs, UK).The DNA digestion mix contained 1 mmol/l Tris-HCl, 0.25 mmol/l MgCl₂ and 1 mmol/l CaCl₂ and 0.4 U DNaseI. DNA was digested for 10 min at 37°C, followed by DNase inactivation for 10 min at 75°C. The final reverse transcription mix contained 25 mmol/l KCL, 10 mmol/l Tris-HCl, 0,6 mmol/l MgCl₂, 2 mmol/l DTT, 4 µmol poly-T or gene specific primer, 0.5 mmol/l of each dNTP and 200 U of BioSkript Moloney Murine Leukemia Virus reverse transcriptase (Bioline, Germany). The entire digestion mixture was incubated with the primer for 5 min at 70°C to enable primer annealing, then cooled on ice. Reverse transcription was carried out for 1 hr at 42°C and the enzyme was subsequently inactivated for 10 min at 70°C. The cDNA including the 3’ terminal region was amplified with the specific primer NOS_seq_3-F3 and a ‘poly-T adapter primer’, that is a polyT primer to which a specific adapter sequence was added (Sambrook and Russell, 2001b) (Supplementary file 1). Subsequently, a nested PCR was performed using a second specific primer (NOS_seq_3-F6) and a primer that contained only the specific adapter sequence of the ‘polyT adapter primer’ to increase PCR specificity (Supplementary file 1, same PCR conditions as above).

The 5’ terminal region of 200 bp was obtained from a full transcriptome sequencing approach of female antennae, which covered the full-length NOS mRNA sequence. RNA sequencing was performed by a commercial service provider (Fasteris, Switzerland), using the HiSeq TM2000 Sequencing System (Illumina, USA) with 100 bp single reads, on 5 μg total RNA isolated from female P. triangulum antennae. CLC Genomics Workbench was used for sequence assembly of the resulting 75 million reads. Reads were quality-trimmed with standard settings and subsequently assembled using the following CLC parameters: nucleotide mismatch cost = 2; insertion cost = 2; deletion cost = 2; length fraction = 0.3; similarity = 0.9. Conflicts among the individual bases were resolved by voting for the base with highest frequency. Contigs shorter than 250 bp were discarded.

To sequence the entire NOS transcript from eggs, cDNA was generated by reverse transcription with a poly-T primer and additionally a specific, central NOS_RT_R1 primer, followed by PCR amplification using various primer combinations to cover the whole transcript sequence (Supplementary file 1). Additionally, the sequence of the 5’ terminal region was confirmed by RT-PCR of mRNA from P. triangulum eggs, using primers NOS_seq_5-F6 and NOS_seq_5-R3 (Supplementary file 1) and subsequent sequencing.

Even though we used a large number of primers to cover the gene, we did not find sections with signals for two different bases at the same site. Thus we infer that there is only one NOS gene in the P. triangulum genome, as in most invertebrates (Labbé et al., 2009). In addition, the transcriptome dataset did not reveal any other transcript that was annotated as nitric oxide synthase.

The GenBank accession numbers for the P. triangulum NOS (Pt-NOS) gene sequence is: KJ425525, for the NOS mRNA of P. triangulum eggs: KJ425526, and for the NOS mRNA in P. triangulum female antennae: KJ425527.

Phylogenetic analysis of NOS gene sequences

NOS coding sequences of 23 insect species from five orders were acquired from the NCBI database. Along with the P. triangulum NOS sequence, these were translated and aligned using Geneious (Version 6.0.5, created by Biomatters, Geneious, New Zealand). The highly variable 5’ end was trimmed. An approximately-maximum-likelihood tree was created with FastTree (Price et al., 2010; Price et al., 2009). Local support values were estimated with the Shimodaira-Hasegawa test based on 1000 samples without re-optimizing the branch lengths for the resampled alignments (Price et al., 2010). Bayesian estimates were made with the program MrBayes 3.1.2 (Huelsenbeck et al., 2001; Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). The MCMC analysis was conducted under a mixed amino acid rate model (prset aamodelpr = mixed). After 1,000,000 generations, with trees sampled every 1000 generations, the standard deviation of split frequencies was consistently lower than 0.01. We discarded the first 100 of the sampled trees (10% burn-in) and computed a 50% majority rule consensus tree with posterior probability values for every node. The trees estimated by both methods were nearly identical, so they were combined into a single figure.

Additional data 1: Bioassay for an antifungal effect of paralyzed honeybees

To test whether paralyzed bees alone show some antifungal activity, we conducted an additional experiment. Petri dishes (N = 12) inoculated with conidia of Aspergillus flavus isolated from brood cells were prepared as described in the main text. Bees from brood cells with two bees were separately placed on the agar. Each of the bees as well as a control area without bee was covered by a cap with a volume of 3 ml (similar to a typical brood cell). Petri dishes were incubated at 25°C in a dark climate chamber. After 24 h fungus growth on the area covered by each of the caps was categorized as: 0 = no fungus, 1 = very little fungus (only few hyphae at the rim), 2 = little fungus (several hyphae at the rim), 3 = strong fungus (area overgrown with hyphae) (Appendix 1—table 1). We tested for overall differences in fungus growth among the three treatment groups using a Friedman test (Software SPSS Version 24). Subsequently, pairwise comparisons were conducted using Bonferroni-corrected Wilcoxon tests.

Petri dish	Bee with egg	Bee without egg	Control
1	0	3	3
2	0	2	2
3	0	2	3
4	1	2	3
5	1	3	3
6	0	3	3
7	0	3	3
8	0	3	3
9	0	3	3
10	0	3	3
11	0	3	3
12	0	3	3

The medians (and quartiles) were 0 (0, 0) for the ‘bee with egg’, 3 (2.25, 3) for the ‘bee without egg’and 3 (3, 3) for the control area without bee. There was a significant overall difference among medians (Friedman test: chi2(tie corrected) = 22.9, D.F. = 2, P = 0.000019). Pairwise comparisons revealed significant differences between the group “bee with egg” and both others (p = 0.0022 in both cases). The groups ‘bee without egg’ and ‘control’ did not differ significantly (p = 1.0). The results show that under the cap with the bee carrying an egg, fungus growth is nearly completely inhibited, whereas the areas with a bee not carrying an egg and the controls did not differ. Thus, there is no evidence that a paralyzed bee produces any antifungal gas or relevant amounts of NO^⋅.

Comparison
Egg/Embalming	Egg/Embalming	Hazard ratio	95% conf. interval
+/+	-/+	0.07	0.19 – 0.03
+/+	-/-	0.13	0.36 – 0.05
+/+	+/-	0.65	0.90 – 0.47
-/+	-/-	0.48	0.79 – 0.29
+/-	-/+	0.61	0.45 – 0.83
+/-	-/-	0.22	0.10 – 0.47

Additional discussion 1

Oxygen content of air is 21% (=210000ppm). Thus, in a brood cell of 3.2 ml there are about 670µl oxygen. Based on (limited) available data on oxygen uptake of Hymenoptera eggs (0.8µl O2/mg/h for a hymenopteran parasitoid egg (Fisher, 1963), a beewolf egg of 5mg would need 4µl O2/h. Living honeybees take up about 0.2µl O2/mg/h (Allen, 1959), thus a honeybee worker of about 80mg will consume 16µl O2/h. Consequently, the oxygen available in a brood cell should be sufficient to support both, metabolism of the egg and oxidation of NO^⋅. Moreover there will be an influx of oxygen form the surrounding sand by diffusion along an emerging gradient. Thus, NO^⋅ is certainly oxidized to NO₂^⋅. in beewolf brood cells. The rate of this conversion can, however, not be specified at the moment.

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Thank you for submitting your article “Nitric oxide radicals are emitted by wasp eggs to kill mold fungi” for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Reviewing Editor and Ian Baldwin as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Your manuscript addresses a very interesting study on how a beewolf preserves the bees that it has collected and protects them from degenerating by the provisioning of NO· by the eggs. All three reviewers provide positive evaluations of your study. Yet, some aspects require further improvement, including an essential additional experiment.

The main issues to be resolved are:

1) The study focuses on NO·, thereby potentially missing other active compounds as highlighted by reviewer 2. To assess the role of NO·, additional evidence to support NO· as being the causal agent is needed, i.e. eliminating NO· with the inhibitor as suggested by reviewer 3. This additional experiment will provide essential information to conclude on the effect of NO· within the total characteristics of the egg.

2) The discussion on the NO· levels in the brood cell, leaking in the soil, survival of larvae and later developmental stages of the wasp, survival of the bees beyond day 3 etc. is not satisfactory. See comments by reviewers 1 and 2. Especially the brief mentioning of mortality of larvae needs more attention and clarification as this is vital for understanding the functioning of NO· as a true resource protectant.

3) The discussion on the NOS gene and its uniqueness in the beewolf. Please clarify what makes the detected NOS gene substantially different from its orthologues and therefore capable of performing a different function (higher NOS production), and better address the option that NO· might arise from different genes.

In addition, the reviewers have made other valuable comments that will be helpful in further improving the manuscript.

Reviewer #1:

This manuscript provides a very interesting study of how beewolf eggs may prevent bee supplies from fungal colonisation by producing high levels of NO· radicals. The study shows that the presence of beewolf eggs prevents mold growth on paralyzed honeybees in the brood cells, that NO· is produced in the eggs, is present around the paralyzed bees and that eggs prevent the growth of a fungus, i.e. Aspergillus flavus. Yet, some important questions remain to fully understand how NO· radicals protect the bees while not harming the beewolf larva or its symbiotic bacteria.

Although the evidence supports the beewolf egg as a source of NO·, the data cannot exclude that NO· is also produced by the paralyzed bee on which the egg has been deposited. The data presented in Figure 2B would support this, as well as the data in Figure 4B. Why was no experiment included in which mold growth on a bee was recorded in the presence of an egg that was presented without a bee substrate to supplement the data in Figure 2B? The data in Figure 4B show that NO· is present on a bee on which an egg has been laid but it cannot be excluded that part of this is produced by the bee.

The manuscript does not present all data and some crucial data are presented as supplementary figures. Why are not all crucial data presented in the main text? For instance (a) the data for the 5 additional fungi are not presented at all (Results, first paragraph), (b) Subsection “Identification of the antifungal volatile”, last paragraph, please show these data, there is no space limitation, (c) Subsection “Identification of the antifungal volatile”, last paragraph: why is the data of the Griese analysis of the headspace samples not presented?, (d) Figures 9 and 10 provide support for the production of NO· in the beewolf egg; the picture (Figure 9) is a combination of two pictures (why?); what is the age of the egg shown in Figure 9 (very important in the context of Figure 10 and Figure 5)? (e) fungus growth inhibition was recorded at 24, 48 and 72 hours (subsection “Fungus inhibition assays”, last paragraph) but no data for 48 or 72 h are presented.

In Figure 6, please provide the exact P value for the comparison between control and D-NAME instead of stating that it is > 0.05.

The picture in Figure 3 presents only one replication. How many replicates were there and how similar were the results? Preferably the pictures of the replicates should be presented in a supplementary figure. The number of replications is low for some experiments, e.g. for timing of NO· emission only 4 replicates were used but for the iodometry experiment a large number of eggs (233) has been used.

Please add a note on the number of replications to the legend of Figure 6 and Figure 10.

The discussion on how the beewolf eggs and the Streptomyces bacteria survive the high NO· levels is not convincing to me. The authors first argue that the NO· concentrations in the brood cell are way too high for fungi to develop resistance (Discussion, twelfth paragraph) but then state that potentially the Streptomyces bacteria are resistant to the NO·, maybe helped by being at the ceiling of the brood cell. However, given the dimensions and the average very high NO· concentration the concentration near the ceiling is likely still very high. Moreover, the arguments given for how the beewolf larvae may survive the high concentrations (Discussion) are not convincing; after all they are the source of the high levels being emitted and even as larva they are killed by these high concentrations.

How do the larvae that emerge from the NO· producing eggs survive in the brood cell? The comment in the twelfth paragraph of the Discussion suggests they do not, which would imply that the NO· concentration drops with time if the larvae are to survive. If that is true (is it?) then how are the bees protected from molding during days 3-11 when the larva develops (Figure 1)? The study provides information on NO· production with time but not on NO· concentration during the 11 days of larval development, which would be needed for this.

The manuscript should provide answers to these questions to be fully convincing on the protective effect of egg-produced high NO· concentrations in terms of preventing their food to be infested by molds while they themselves should survive the intoxicated brood cell.

Reviewer #2:

In this interesting manuscript, Strohm et al. propose a new mechanism of brood defense from fungal pathogens based on the production of antifungal volatile compounds. This is a novel and potentially important idea that offers new insights into how insects might combat pathogens during vulnerable life cycle stages. On the whole, I enjoyed reading this well-written manuscript and the ideas that it contains. However, I was left with some questions that I hope the authors can address:

1) Given the existence of other defense mechanisms in the beewolf system, is there any potential for NO production to interact synergistically or antagonistically with these other defense mechanisms? For example, are there any compounds on the eggs themselves that might provide some antifungal activity? A broader discussion (e.g., in the Introduction) of the various beewolf defense mechanisms and their importance in different phases of the beewolf life cycle might help clarify these issues. This issue also intersects with the discussion of NO resistance by the Streptomyces symbiont, such that there might be trade-offs between defenses active at different life cycle stages.

2) The author’s focus on NO is quite directed, and so might cause them to miss other potential volatile compounds that are also involved in the observed bioactivities. Better justifying this approach would be valuable. There also seems to be some disconnect between the first paragraph of the subsection “Identification of the antifungal volatile”, where the authors seem to interpret the odor given off by the beewolf eggs as similar to NO₂, and the first paragraph of the subsection “Identification of the antimicrobial volatile”, where samples where nitrate was reduced to detect NO₂ did not differ from those that were not reduced, implying that only NO (and not NO₂) was produced by the eggs, despite the odor.

3) The authors’ calculations of likely NO concentrations in brood cells is very interesting, but I am unsure how likely these values are to relate to the actual values in the wild. In particular, how likely is it that there is “no loss due to reactions or leaking out of the confined space of brood cells”? It seems that soil could conceivably be quite porous and so allow NO to escape the brood cell. A pulse that diffuses quickly would impact the need for and nature of NO resistance mechanisms that the authors discuss extensively. Similarly, what is the likely soil temperature of a typical beewolf brood cell? The assay temperatures strike me as being warmer than many soils in temperate Europe, although I imagine that the insect could select appropriate sites to meet this constraint. Finally, the authors propose oxic mechanisms for NO (bio)chemistry (e.g., conversion of NO to NO₂) without specifying if the brood cells are oxic or anoxic. Reduced oxygen in brood cells seems plausible if they are sealed while the egg is developing (and presumably respiring).

4) The authors give several potential mechanisms for NO resistance, but it is unclear how plausible these are given the potentially high concentrations of NO that might be present. A more quantitative discussion of NO resistance would be helpful here, e.g., are the proposed resistance mechanisms likely to be sufficient under the proposed levels of NO accumulation?

5) Discussion, twelfth paragraph: the authors propose that coating the symbiotic Streptomyces in hydrocarbons might protect them from NO toxicity. However, the bees are also coated in hydrocarbons (Introduction, fourth paragraph). Wouldn’t the resistance mechanism proposed for the bacteria then also protect fungi growing on the bees? Antioxidants are also proposed to be mixed with these hydrocarbons – how likely are these to exist?

6) Discussion, fifteenth paragraph: the observation that larvae are not resistant to NO produced by the eggs is concerning. Are other life cycle stages similarly sensitive to NO, e.g., eggs exposed before they start producing NO or immediately after? The sensitivity of larvae might imply that NO does not accumulate in the brood cells for very long (see also my comment #3 above).

7) I am not entirely clear how to interpret Figure 9 because I am unclear what baseline level of staining to expect before NOS induction. Comparisons between time points and/or other species where NOS is not thought to be induced would provide useful controls for this experiment.

8) The focus on the detected NOS gene as being responsible for the observed NOS activity makes the argument that this gene behaves differently compared to all other known genes without direct evidence for such divergent behavior. The one inferred splice variant by itself seems to provide weak evidence for higher activity, given that higher NOS activity observed in humans is due to different genes (vs. splice variants of the same gene) and that NOS activity in humans is of a different order of magnitude than implied here for beewolves. The inhibition experiment provides some evidence for NOS being involved, although there is still the assumption that NOS is the only enzyme affected by the inhibitor. Differential gene expression matching NO production, direct assays of the NOS variants, or even signatures of adaptive evolution (e.g., Dn/Ds metrics) would provide more convincing support for the uniqueness of the beewolf NOS, especially when combined with explicit comparisons to related species that due not produce similarly high levels of NO as controls.

Reviewer #3:

This is a very nice paper that touches on different fascinating aspects of insect’s biology, from an ecological to a molecular point of view, and should interest a broad audience.

This study shows that eggs of the parasitic wasp Philanthus triangulum emit high amounts of nitric oxide as antifungal compounds to protect themselves and preserve the food source of their progeny. The enzyme responsible for NO production is identified (NOS) and a differential splicing event between egg and adult forms may explain the high levels observed in eggs. These are novel and quite interesting findings that complement other defense strategies that have been previously reported in insects. The story is well written and nicely presented. Data are convincing. The Discussion is complete and carefully addresses the novelty and the biochemical and biological implications of the findings.

That eggs emit a volatile compound that has antifungal activity is demonstrated by 1) showing that host bees not in direct or indirect contact with P. triangulum eggs get more infected with mold fungi, 2) showing that eggs release an antifungal volatile when placed above a culture of different fungal strains growing on agar plates. That eggs emit NO is demonstrated by histochemical and colorimetric methods and by using fluorescent probes. However, that egg-released NO is responsible for inhibiting fungal growth is only supported by the correlation between the above observations and by previous reports on the mycotic activity of NO. I see the difficulty to genetically prove the causality of NO production in the antifungal effect of egg-derived volatile production. A simple additional experiment may however help to strengthen this hypothesis. It would be important to inhibit NO release in eggs by using L-NAME, the NOS-inhibiting substance used for Figure 6, and to show that egg-induced inhibition of fungal growth on agar plate (as in Figure 3) is abolished or greatly diminished.

[Editors’ note: further revisions were requested prior to acceptance, as described below.]

Thank you for submitting your article “Nitric oxide radicals are emitted by wasp eggs to kill mold fungi” for consideration by eLife. Your article has been reviewed by a Reviewing Editor and Ian Baldwin as the Senior Editor.

In response to the reviews you have carried out an additional experiment and have modified the manuscript in various places. As a result the manuscript has been clearly improved. Yet, a few issues still remain.

The additional experiment tests the effect of synthetic NO on fungal infection of honeybees and shows that synthetic NO protects the bees from infection. This experiment was done for logistic reasons as an alternative to the suggested experiment (eliminating NO· from the natural mixture with the inhibitor). The result underlines the involvement of NO but leaves the option of other compounds being involved open. One of the reviewers had commented that the focus on NO is quite directed, and so might result in missing other potential volatile compounds that are also involved in the observed bioactivities. In response to the question to better justify the focus on NO you comment that is not reasonable. If the suggested experiment had been done that might be the case but because the involvement of other compounds has not been excluded I consider it reasonable to explain why you focussed on NO.

In response to the comment that “the observation that larvae are not resistant to NO produced by the eggs is concerning.” you respond with comments in the response letter but this has not resulted in a change in the manuscript. It will be important to provide information in the manuscript itself why the larvae will not suffer from NO as this is vital to the understanding of the protective activity of the NO in conserving a resource. This aspect was also identified by one of the other reviewers.

https://doi.org/10.7554/eLife.43718.055

Your manuscript addresses a very interesting study on how a beewolf preserves the bees that it has collected and protects them from degenerating by the provisioning of NO· by the eggs. All three reviewers provide positive evaluations of your study. Yet, some aspects require further improvement, including an essential additional experiment.

The main issues to be resolved are:

1) The study focuses on NO·, thereby potentially missing other active compounds as highlighted by reviewer 2. To assess the role of NO·, additional evidence to support NO· as being the causal agent is needed, i.e. eliminating NO· with the inhibitor as suggested by reviewer 3. This additional experiment will provide essential information to conclude on the effect of NO· within the total characteristics of the egg.

We have conducted an additional experiment to assess whether NO can be responsible for the strong antifungal effect of the gas emitted by beewolf eggs. Since we do not have beewolves available during winter and spring due to the obligatory diapause of this insect species, we were not able to do the inhibitory experiment as suggested by reviewer 3. Instead, we applied synthetic NO to artificial brood cells with bees in amounts that mimicked the estimated concentrations in natural brood cells. There was a significant delay of fungus growth on the bees that were subjected to the NO compared to respective controls. Moreover, comparison of the hazard ratio (the appropriate measure of effect size for such survival analyses) calculated for this experiment and a similar experiment that was previously conducted with beewolf eggs instead of synthetic NO revealed similar values with mutually overlapping 95% confidence intervals. (side note: It is not meaningful to compare the data of the two experiments directly, since they were conducted at different times with different bees and possibly different strains of fungi; therefore we compared the effect sizes obtained in the two experiments). Thus, the experiment shows equivalent antifungal effects of artificial and beewolf-produced nitric oxide in situ, and there was no evidence for an additional agent being active in the gas emitted by beewolf eggs.

2) The discussion on the NO· levels in the brood cell, leaking in the soil, survival of larvae and later developmental stages of the wasp, survival of the bees beyond day 3 etc. is not satisfactory. See comments by reviewers 1 and 2. Especially the brief mentioning of mortality of larvae needs more attention and clarification as this is vital for understanding the functioning of NO· as a true resource protectant.

Please see our comments below.

3) The discussion on the NOS gene and its uniqueness in the beewolf. Please clarify what makes the detected NOS gene substantially different from its orthologues and therefore capable of performing a different function (higher NOS production), and better address the option that NO· might arise from different genes.

Please see our comments below.

In addition, the reviewers have made other valuable comments that will be helpful in further improving the manuscript.

Reviewer #1:

This manuscript provides a very interesting study of how beewolf eggs may prevent bee supplies from fungal colonisation by producing high levels of NO· radicals. The study shows that the presence of beewolf eggs prevents mold growth on paralyzed honeybees in the brood cells, that NO· is produced in the eggs, is present around the paralyzed bees and that eggs prevent the growth of a fungus, i.e. Aspergillus flavus. Yet, some important questions remain to fully understand how NO· radicals protect the bees while not harming the beewolf larva or its symbiotic bacteria.

Although the evidence supports the beewolf egg as a source of NO·, the data cannot exclude that NO· is also produced by the paralyzed bee on which the egg has been deposited. The data presented in Figure 2B would support this, as well as the data in Figure 4B. Why was no experiment included in which mold growth on a bee was recorded in the presence of an egg that was presented without a bee substrate to supplement the data in Figure 2B? The data in Figure 4B show that NO· is present on a bee on which an egg has been laid but it cannot be excluded that part of this is produced by the bee.

We indeed cannot completely rule out the possibility that paralyzed honeybees emit small amounts of nitric oxide. However, we added the results of an experiment that shows that a paralyzed honeybee does not delay fungus growth and thus does not produce relevant amounts of NO. We present this experiment in the SI section (SI Additional data 1) in order not to overload the main text. Thus, our results show that (i) beewolf eggs produce incredibly high concentrations of NO, while (ii) NO emission from paralyzed bees (if it indeed occurs at all, which seems very unlikely given that NO emission has never been shown previously for an adult insect) are ecologically negligible.

The manuscript does not present all data and some crucial data are presented as supplementary figures. Why are not all crucial data presented in the main text? For instance a) the data for the 5 additional fungi are not presented at all (Results, first paragraph).

It is stated in the text that for the bioassays with 5 fungi (N=8 replicates each), in all cases there was a clear difference with no fungus growing in the area exposed to the gas emanating from the egg and fungus growth in the control area (as exemplified in Figure 3). Unfortunately, we have not taken photos of all these experiments, since the results were so clear-cut. We have changed the text to clarify this. Concerning other figures, we opted not to overload the main text with figures, so we left additional data supporting – but not directly critical for – the main points of the study in the supplementary data.

b) Subsection “Identification of the antifungal volatile”, last paragraph, please show these data, there is no space limitation.

Since pictures of the eggs are presented, only a picture of an injected larva is not shown. We initially did not add these, because they are somewhat blurred due to the larvae moving during exposure. Anyhow, we have now added pictures of a larva to the SI file. Moreover, we provide all pictures of all eggs and larvae injected with the fluorescent probe as a source data file.

c) Subsection “Identification of the antifungal volatile”, last paragraph: why is the data of the Griese analysis of the headspace samples not presented?

We are not sure what the reviewer means here. The Griess assay as employed here was to prove the existence of nitrite that can, with the setup that we applied only be generated by a gas, either NO or NO₂, emitted from the egg. We have now added an original picture of vials showing the positive reaction. We did not use the Griess reaction for quantification, since this assay is not suitable for prolonged sampling as would be necessary for the analysis of the emission from beewolf eggs. The picture (Figure 4—figure supplement 1) is not very appealing since it was meant for documentation, not for publication, but it clearly shows the color change in the Griess reagent exposed to the headspace of beewolf eggs.

d) Figures 9 and 10 provide support for the production of NO· in the beewolf egg. The picture (Figure 9) is a combination of two pictures (why?).

The egg was too large to fit into the field of view of the camera and we did not have a lower magnification lens available, so we had to take two pictures (basically the left and the right part) and stitched these. This is stated more clearly now.

What is the age of the egg shown in Figure 9 (very important in the context of Figure 10 and Figure 5)?

The egg was fixed 15-16h after oviposition, the time of peak NO production. This is stated now in the revised manuscript.

e) fungus growth inhibition was recorded at 24, 48 and 72 hours (subsection “Fungus inhibition assays”, last paragraph) but no data for 48 or 72 h are presented.

We suspect that the line numbers the reviewer refers to differ from the PDF that we provided (which is the same as the one that can be downloaded from the eLife site). So we assume that the reviewer refers to the last paragraph of the subsection “Fungus inhibition assays”, It is stated that the results for 48 and 72 h are the same as for 24 h. We have changed the text to clarify this.

In Figure 6, please provide the exact P value for the comparison between control and D-NAME instead of stating that it is > 0.05.

The P value was added to the figure.

The picture in Figure 3 presents only one replication. How many replicates were there and how similar were the results? Preferably the pictures of the replicates should be presented in a supplementary figure.

The number of replicates for the Aspergillus isolates from brood cells (N = 20) and for the five additional fungi (N=8) were given in the text. Unfortunately, as mentioned above, we did not take pictures of all the petri dishes of these experiments. The results were clear-cut, with no exceptions and no intermediate cases. This is also stated in the Materials and methods, but is now better explained in the revised text.

The number of replications is low for some experiments, e.g. for timing of NO· emission only 4 replicates were used but for the iodometry experiment a large number of eggs (233) has been used.

As stated in the paragraph “General experimental procedures” in the Materials and methods section, availability of beewolf eggs is often limited and difficult to predict. It is particularly challenging to obtain large sample sizes, if the time of oviposition has to be known and the egg has to be collected shortly thereafter, as is the case for the experiment to reveal the timing of NO emission. Beewolves lay their eggs mostly during the night, thus monitoring oviposition is rather laborious. We managed to measure 4 replicates for the whole period (0-22h), these were very similar. We did several additional measurements of other eggs that did, however, not span the whole period. We have now included all these measurements and modified the figure accordingly.

Knowing that NO production within the first hours after oviposition is rather low, it was comparatively much easier to produce a large sample for the quantification of NO emission.

Please add a note on the number of replications to the legend of Figure 6 and Figure 10.

We added the sample sizes to the legend of Figure 6.

The discussion on how the beewolf eggs and the Streptomyces bacteria survive the high NO· levels is not convincing to me. The authors first argue that the NO· concentrations in the brood cell are way too high for fungi to develop resistance (Discussion, twelfth paragraph) but then state that potentially the Streptomyces bacteria are resistant to the NO·, maybe helped by being at the ceiling of the brood cell. However, given the dimensions and the average very high NO· concentration the concentration near the ceiling is likely still very high.

We agree that the survival of both, the eggs and the bacteria are surprising taking into account the high concentrations of nitrogen oxides. At the moment we can only speculate about the ultimate causes and the proximate mechanisms.

With regard to the eggs: As has been stated in the manuscript, there might be no “free” NO in the egg tissue. Possibly, the NO is bound to carrier molecules that take them to the egg shell for release. After oxidation to NO₂, diffusibility is considerably reduced compared to NO so that NO₂ might not be able to penetrate the egg shell. The larva however, has only a very thin cuticle and gas exchange happens by means of tracheae. Thus NO₂ might enter easily and kill the larva. In addition, the eggs likely mount countermeasures to protect itself against NO (e.g. controlling reactive oxygen species that in situ drastically enhance the detrimental effect of NO), while the larvae won’t do this under natural conditions.

With regard to the symbionts, one important ultimate aspect that was not made clear enough in the manuscript is that the symbiotic bacteria coevolved with beewolves; meaning the same bacterial lineages were repeatedly exposed to NO and were thus subject to strong selection. The fungi on the other hand that are to be found in beewolf brood cells are unspecialized ubiquitous and opportunistic. Thus there is no repeated exposition of the same fungal strains to high concentrations of NO. Consequently, there is negligible selection for resistance.

There might be several coactive mechanisms that protect the bacteria: elevated location in the brood cell, screening by the lipids of the surrounding matrix, antioxidants in the surrounding matrix, molecular detoxification of NO and/or NO₂. These aspects have been discussed more explicitly and carefully in the revised manuscript, and future experiments will be targeted towards elucidating the resistance of both beewolf and symbionts against NO.

Moreover, the arguments given for how the beewolf larvae may survive the high concentrations (Discussion) are not convincing; after all they are the source of the high levels being emitted and even as larva they are killed by these high concentrations.

We suspect that the reviewer does not refer to larvae but to eggs as producers of NO here, so please see our response to the preceding comment.

How do the larvae that emerge from the NO· producing eggs survive in the brood cell? The comment in the twelfth paragraph of the Discussion suggests they do not, which would imply that the NO· concentration drops with time if the larvae are to survive.

Since the brood cell is constructed in sandy soil, there is certainly a gradual loss of nitrogen oxides due to diffusion out of the brood cell and dissolution in water. However, this leakage of nitrogen oxides is most probably slow, so that during peak NO production the concentrations will be high. Actually, the typical smell of NO₂ disappears by the time the larvae hatch, so larvae are not exposed to the nitrogen oxides (at least not to such high concentrations).

If that is true (is it?) then how are the bees protected from molding during days 3-11 when the larva develops (Figure 1)?

As is stated in the manuscript, the brood cell fumigation considerably reduces the detrimental effect of fast growing mold fungi by killing them. Thus the major threat to the larvae is eliminated. Spores that germinate after the nitrogen oxide levels have dropped or are not exposed to the gas because they are covered by the bee’s body might be able to grow and compete with the larvae. As is shown by the added experiment, embalming of the bees corroborates the antifungal effect of fumigation. However, even the combined protective effect of the fumigation and the embalming is, of course, not perfect. In fact, we have already reported that cocoons from brood cells that show some fungus infestation are smaller than cocoons from not infested brood cells (Strohm, 2000). However, in most cases, larvae will be able to complete feeding and spin their cocoons. Then the antibiotics on the cocoon walls that are produced by the symbiotic Streptomyces bacteria will take over the protection of the larvae.

The study provides information on NO· production with time but not on NO· concentration during the 11 days of larval development, which would be needed for this.

It is very difficult to measure NO over time on such a small spatial scale in situ. Although the concentrations in the brood cell are high, the absolute amounts are small. There are devices to measure NO in tissue but, according to the manufacturers these only work well in aqueous surroundings. So, yes, unfortunately, we do not have data on the NO concentration in the brood cell over time. As stated above, however, the smell of NO₂ disappears before larvae hatch. Hence, it appears that the short burst of NO production serves to kill fungi, and the NO then diffuses slowly out of the brood cell, leaving a sanitized environment that allows the larva to hatch successfully.

The manuscript should provide answers to these questions to be fully convincing on the protective effect of egg-produced high NO· concentrations in terms of preventing their food to be infested by molds while they themselves should survive the intoxicated brood cell.

We believe that the manuscript provides a thorough description of a novel antimicrobial strategy in a ground-developing insect, showing that the production of high concentrations of NO reduces fungal infestation of the beewolf’s provisions in vitro and in situ. We have added additional data supporting the sanitizing role of NO in situ, and expanded on the discussion of how the beewolf egg and the symbiotic bacteria survive NO exposure in the revised manuscript.

Reviewer #2:

In this interesting manuscript, Strohm et al. propose a new mechanism of brood defense from fungal pathogens based on the production of antifungal volatile compounds. This is a novel and potentially important idea that offers new insights into how insects might combat pathogens during vulnerable life cycle stages. On the whole, I enjoyed reading this well-written manuscript and the ideas that it contains. However, I was left with some questions that I hope the authors can address:

1) Given the existence of other defense mechanisms in the beewolf system, is there any potential for NO production to interact synergistically or antagonistically with these other defense mechanisms? For example, are there any compounds on the eggs themselves that might provide some antifungal activity? A broader discussion (e.g., in the Introduction) of the various beewolf defense mechanisms and their importance in different phases of the beewolf life cycle might help clarify these issues. This issue also intersects with the discussion of NO resistance by the Streptomyces symbiont, such that there might be trade-offs between defenses active at different life cycle stages.

We already had data available on the possible interaction of the fumigation with the embalming of the honeybees with hydrocarbons. We had not presented these data in this manuscript because we believed it to be beyond the scope of this study. However, because of the reviewer’s comment, we added these data to the revised manuscript. In fact, embalming and fumigation together have a stronger antifungal effect than either mechanism alone. The interaction with the symbiotic bacteria is of course extremely interesting and is currently under investigation. We added a brief description of the antimicrobial symbiosis of beewolves to the Introduction.

2) The author’s focus on NO is quite directed, and so might cause them to miss other potential volatile compounds that are also involved in the observed bioactivities. Better justifying this approach would be valuable.

There are some other strong gaseous oxidants. However, as a result of many preliminary approaches and considerations, NO was, by far, the most plausible alternative. We do think that a detailed justification of the focus on NO is not reasonable.

There also seems to be some disconnect between the first paragraph of the subsection “Identification of the antifungal volatile”, where the authors seem to interpret the odor given off by the beewolf eggs as similar to NO₂, and the first paragraph of the subsection “Identification of the antimicrobial volatile”, where samples where nitrate was reduced to detect NO₂ did not differ from those that were not reduced, implying that only NO (and not NO₂) was produced by the eggs, despite the odor.

The odor definitely is caused by NO₂; as stated, NO is odorless (though being highly reactive, it has less oxidative power than NO₂). The Griess assay resulted in red coloration both with and without reduction. The statement of no difference between the groups meant that both showed the red coloration. Because the red azo dye that is formed is not perfectly stable over time, as stated by the manufacturer’s instructions of the kit, quantitative measurements for extended exposure times are not recommended. This is now stated in the revised version.

3) The authors’ calculations of likely NO concentrations in brood cells is very interesting, but I am unsure how likely these values are to relate to the actual values in the wild. In particular, how likely is it that there is “no loss due to reactions or leaking out of the confined space of brood cells”? It seems that soil could conceivably be quite porous and so allow NO to escape the brood cell. A pulse that diffuses quickly would impact the need for and nature of NO resistance mechanisms that the authors discuss extensively.

As stated above, we unfortunately have no actual data on the time course of NO/NO₂ concentrations in the brood cells. We discuss the issue of leakage in more detail now. Beewolves construct their brood cells in rather compact sandy soil. Moreover, the walls of the nest burrows and the brood cells are covered with a layer of hydrocarbons that might provide an additional barrier; this is now mentioned in the revised text. So brood cells are neither perfectly sealed nor are they porous enough to let gases leak out easily. Because there is no airflow within the brood cell, removal of NO/NO₂ will happen by diffusion only. Since most NO is produced within a short 2h burst, it will accumulate to high concentrations that are detrimental to microbes. The time course of diffusion into the surrounding soil remains thus far unknown und hard to quantify.

Similarly, what is the likely soil temperature of a typical beewolf brood cell? The assay temperatures strike me as being warmer than many soils in temperate Europe, although I imagine that the insect could select appropriate sites to meet this constraint.

Of course temperatures in the field vary somewhat due to differences in depth of the brood cells, sand structure, sand moisture, inclination and orientation of the nest site. Beewolves establish their nests only at very sunny sites and sandy soil and the incubation temperatures used in the experiments reflect a representative temperature of natural brood cells {Herzner and Strohm, 2008). Moreover, larval development is best around 25-30°C (Figure 5 in Strohm, 2000). This information has now been added to the revised version.

Finally, the authors propose oxic mechanisms for NO (bio)chemistry (e.g., conversion of NO to NO₂) without specifying if the brood cells are oxic or anoxic. Reduced oxygen in brood cells seems plausible if they are sealed while the egg is developing (and presumably respiring).

This issue is discussed in more detail now in the SI file.

4) The authors give several potential mechanisms for NO resistance, but it is unclear how plausible these are given the potentially high concentrations of NO that might be present. A more quantitative discussion of NO resistance would be helpful here, e.g., are the proposed resistance mechanisms likely to be sufficient under the proposed levels of NO accumulation?

We are not sure whether the reviewer refers to a possible resistance of the symbiotic bacteria or the beewolf egg itself.

With regard to the bacteria we changed the text to make clear that at the moment we can only speculate about possible mechanisms and added some references. Since a more quantitative analysis is experimentally also elaborate, the detailed reaction of the symbiotic bacteria will be the focus of a future study.

With regard to beewolf eggs, we mention nitrophorins that might carry the NO to the egg shell, thus little “free” NO would occur in the tissue of the embryo. As stated in the text, such nitrophorins are known from blood sucking bugs. These nitrophorins might be produced in amounts sufficient for the transport of NO. However, we do not find it meaningful to further speculate about this and other mechanisms. Investigation of these mechanisms goes far beyond the scope of the present manuscript.

5) Discussion, twelfth paragraph: the authors propose that coating the symbiotic Streptomyces in hydrocarbons might protect them from NO toxicity. However, the bees are also coated in hydrocarbons (Introduction, fourth paragraph). Wouldn’t the resistance mechanism proposed for the bacteria then also protect fungi growing on the bees? Antioxidants are also proposed to be mixed with these hydrocarbons – how likely are these to exist?

Spores embedded in the CHC layer on the bees might indeed be protected against NO. However, these would also be shielded from liquid water that would be necessary for germination, so these would not be dangerous for beewolf progeny. Spores that are not fully covered by hydrocarbons might germinate but will likely be affected by the NO.

With regard to the antioxidants, we have preliminary data that antioxidants are present in the matrix surrounding the symbionts in the brood cell. However, this is again beyond the scope of the current manuscript and needs substantial additional experimental validation.

6) Discussion, fifteenth paragraph: the observation that larvae are not resistant to NO produced by the eggs is concerning. Are other life cycle stages similarly sensitive to NO, e.g., eggs exposed before they start producing NO or immediately after? The sensitivity of larvae might imply that NO does not accumulate in the brood cells for very long (see also my comment #3 above).

As stated above nitrogen oxides will slowly disappear from the brood cells, thus, larvae are not harmed under natural conditions. The mentioned cases of larvae dying from exposition to NO/NO₂ accidentally occurred during early experiments to identify the gas.

We have not tested whether early egg stages are sensitive against NO/NO₂.

7) I am not entirely clear how to interpret Figure 9 because I am unclear what baseline level of staining to expect before NOS induction. Comparisons between time points and/or other species where NOS is not thought to be induced would provide useful controls for this experiment.

Since the method is rather complicated we analyzed only few eggs in this way. According to the literature, without fixation, several enzymes show NADPH diaphorase activity. However, with fixation, blue staining indicates sites of NOS activity. Since the embryonic tissue of this egg (about 15h after oviposition, i.e. during the time of peak NO production) is clearly stained blue, this provides clear evidence for NOS activity.

8) The focus on the detected NOS gene as being responsible for the observed NOS activity makes the argument that this gene behaves differently compared to all other known genes without direct evidence for such divergent behavior. The one inferred splice variant by itself seems to provide weak evidence for higher activity, given that higher NOS activity observed in humans is due to different genes (vs. splice variants of the same gene) and that NOS activity in humans is of a different order of magnitude than implied here for beewolves. The inhibition experiment provides some evidence for NOS being involved, although there is still the assumption that NOS is the only enzyme affected by the inhibitor. Differential gene expression matching NO production, direct assays of the NOS variants, or even signatures of adaptive evolution (e.g., Dn/Ds metrics) would provide more convincing support for the uniqueness of the beewolf NOS, especially when combined with explicit comparisons to related species that due not produce similarly high levels of NO as controls.

We agree that there might have been too much focus on the NOS gene and the alternative splicing as an explanation of the elevated NO production rates. We have now changed the text to be more cautious, stated that there might be another, yet unknown pathway and conclude with the hypothesis that the NOS is an important component of NO production in beewolf eggs. However, we have now obtained a draft genome sequence of P. triangulum, and there is only one Pt-NOS gene, confirming our finding from transcriptomic analyses. The genome is currently subject to careful annotation and evolutionary analyses. In addition, gene expression analyses are included in the manuscript (Figure 10, Figure 7 in the revised manuscript), and the peak in NOS transcripts corresponds to the peak in NO production at the respective temperature, providing strong support for the production of NO from the single NOS that occurs in beewolves.

Reviewer #3:

This is a very nice paper that touches on different fascinating aspects of insect’s biology, from an ecological to a molecular point of view, and should interest a broad audience.

This study shows that eggs of the parasitic wasp Philanthus triangulum emit high amounts of nitric oxide as antifungal compounds to protect themselves and preserve the food source of their progeny. The enzyme responsible for NO production is identified (NOS) and a differential splicing event between egg and adult forms may explain the high levels observed in eggs. These are novel and quite interesting findings that complement other defense strategies that have been previously reported in insects. The story is well written and nicely presented. Data are convincing. The Discussion is complete and carefully addresses the novelty and the biochemical and biological implications of the findings.

That eggs emit a volatile compound that has antifungal activity is demonstrated by 1) showing that host bees not in direct or indirect contact with P. triangulum eggs get more infected with mold fungi, 2) showing that eggs release an antifungal volatile when placed above a culture of different fungal strains growing on agar plates. That eggs emit NO is demonstrated by histochemical and colorimetric methods and by using fluorescent probes. However, that egg-released NO is responsible for inhibiting fungal growth is only supported by the correlation between the above observations and by previous reports on the mycotic activity of NO. I see the difficulty to genetically prove the causality of NO production in the antifungal effect of egg-derived volatile production. A simple additional experiment may however help to strengthen this hypothesis. It would be important to inhibit NO release in eggs by using L-NAME, the NOS-inhibiting substance used for Figure 6, and to show that egg-induced inhibition of fungal growth on agar plate (as in Figure 3) is abolished or greatly diminished.

As stated above, we have conducted an alternative additional experiment that shows that synthetic NO in concentrations similar to those estimated for brood cells exert a similar effect on fungus growth as the gas emitted by eggs.

[Editors’ note: further revisions were requested prior to acceptance, as described below.]

In response to the reviews you have carried out an additional experiment and have modified the manuscript in various places. As a result the manuscript has been clearly improved. Yet, a few issues still remain.

The additional experiment tests the effect of synthetic NO on fungal infection of honeybees and shows that synthetic NO protects the bees from infection. This experiment was done for logistic reasons as an alternative to the suggested experiment (eliminating NO· from the natural mixture with the inhibitor). The result underlines the involvement of NO but leaves the option of other compounds being involved open. One of the reviewers had commented that the focus on NO is quite directed, and so might result in missing other potential volatile compounds that are also involved in the observed bioactivities. In response to the question to better justify the focus on NO you comment that is not reasonable. If the suggested experiment had been done that might be the case but because the involvement of other compounds has not been excluded I consider it reasonable to explain why you focussed on NO.

We have now added information about the considerations that led us to focus on NO/NO₂. Besides the typical odor of oxidizing gases, we tested the occurrence of an oxidant using iodine/starch solution. The positive reaction clearly showed that there was a strong oxidant. Most of the possible candidates were rather unlikely since they were –to our knowledge- not known to be produced by organisms (O₃) or their occurrence was restricted to special processes (Cl₂). Since NO was known as a ubiquitous biological effector and its release would generate the typical odor (due to its oxidation to NO₂), we focused on NO and conducted the relevant analyses, including the specific Griess assay. Our additional experiment shows that concentrations of synthetic NO similar to natural brood cells have an antimycotic effect that does not deviate from that of the gas emitted by the beewolf eggs. Nevertheless, we cannot exclude that the egg releases small amounts of other volatiles. This is stated now.

In response to the comment that “the observation that larvae are not resistant to NO produced by the eggs is concerning.” you respond with comments in the response letter but this has not resulted in a change in the manuscript. It will be important to provide information in the manuscript itself why the larvae will not suffer from NO as this is vital to the understanding of the protective activity of the NO in conserving a resource. This aspect was also identified by one of the other reviewers.

Actually, we had made some changes, however we have now rephrased the text to more explicitly explain, why larvae do not suffer from the high concentrations of NO that are generated by the eggs.

https://doi.org/10.7554/eLife.43718.056

Author details

1. Erhard Strohm

   Evolutionary Ecology Group, Institute of Zoology, University of Regensburg, Regensburg, Germany
   

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing
   

   For correspondence

   erhard.strohm@ur.de
   

   Competing interests

   No competing interests declared
   

   
   “This ORCID iD identifies the author of this article:”
   0000-0002-8899-9765
   
   

2. Gudrun Herzner

   Evolutionary Ecology Group, Institute of Zoology, University of Regensburg, Regensburg, Germany
   

   Contribution

   Resources, Investigation, Methodology, Writing—review and editing
   

   Competing interests

   No competing interests declared
   

3. Joachim Ruther

   Chemical Ecology Group, Institute of Zoology, University of Regensburg, Regensburg, Germany
   

   Contribution

   Conceptualization, Investigation, Methodology, Writing—review and editing
   

   Competing interests

   No competing interests declared
   

4. Martin Kaltenpoth

   1. Evolutionary Ecology Group, Institute of Zoology, University of Regensburg, Regensburg, Germany
   2. Insect Symbiosis Research Group, Max Planck Institute for Chemical Ecology, Jena, Germany

   Present address

   Evolutionary Ecology, Institute of Organismic and Molecular Evolution, Johannes Gutenberg University, Mainz, Germany
   

   Contribution

   Conceptualization, Resources, Investigation, Methodology, Writing—review and editing
   

   Competing interests

   No competing interests declared
   

   
   “This ORCID iD identifies the author of this article:”
   0000-0001-9450-0345
   
   

5. Tobias Engl

   1. Evolutionary Ecology Group, Institute of Zoology, University of Regensburg, Regensburg, Germany
   2. Insect Symbiosis Research Group, Max Planck Institute for Chemical Ecology, Jena, Germany

   Present address

   Evolutionary Ecology, Institute of Organismic and Molecular Evolution, Johannes Gutenberg University, Mainz, Germany
   

   Contribution

   Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing—review and editing
   

   Competing interests

   No competing interests declared
   

   
   “This ORCID iD identifies the author of this article:”
   0000-0002-2200-2678
   
   

Funding

No external funding was received for this study.

Acknowledgements

We are grateful to Wilhelm Boland for advice, to Clarissa Schill and Nathalie Moske for technical assistance, to Stephan Schneuwly for providing the microinjection device and, in particular, to Jon Seger, Jeremy Field and to the anonymous reviewers for valuable comments on earlier drafts of the manuscript.

Senior Editor

1. Ian T Baldwin, Max Planck Institute for Chemical Ecology, Germany

Reviewing Editor

1. Marcel Dicke, Wageningen University, Netherlands

Publication history

1. Received: November 16, 2018
2. Accepted: May 5, 2019
3. Version of Record published: June 11, 2019 (version 1)

Copyright

© 2019, Strohm et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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