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.

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).

Figure 1

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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.

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^⋅.

The GenBank accession numbers for the P. triangulum NOS (Pt-NOS) gene sequence is: KJ425525, for the NOS mRNA of P. triangulum eggs: KJ425526, for the NOS mRNA in P. triangulum female antennae: KJ425527, for the transcriptome: Bioproject accession number PRJNA542283 with biosample accession number SAMN11793601. Other data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, 5, 6, 6—supplement 1, 7, 8, 10, 11.

1. 1. Strohm E
   2. Herzner G
   3. Ruther J
   4. Kaltenpoth M
   5. Engl T

   (2015) GenBank

   ID KJ425525.1. Philanthus triangulum nitric oxide synthase gene, complete cds, alternatively spliced.

   https://www.ncbi.nlm.nih.gov/nuccore/KJ425525
2. 1. Strohm E
   2. Herzner G
   3. Ruther J
   4. Kaltenpoth M
   5. Engl T

   (2015) GenBank

   ID KJ425526.1. Philanthus triangulum nitric oxide synthase egg isoform mRNA, complete cds, alternatively spliced.

   https://www.ncbi.nlm.nih.gov/nuccore/KJ425526
3. 1. Strohm E
   2. Herzner G
   3. Ruther J
   4. Kaltenpoth M
   5. Engl T

   (2015) GenBank

   ID KJ425527.1. Philanthus triangulum nitric oxide synthase adult isoform mRNA, complete cds, alternatively spliced.

   https://www.ncbi.nlm.nih.gov/nuccore/KJ425527
4. 1. Sandoval-Calderón M
   2. Nechitaylo T
   3. Engl T
   4. Vogel H
   5. Koehler S
   6. Kaltenpoth M

   (2019) Genbank

   ID PRJNA542283. RNAseq reads from Philanthus triangulum and its symbiont, 'Candidatus Streptomyces philanthi'.

   https://www.ncbi.nlm.nih.gov/bioproject/542283
5. 1. Sandoval-Calderón M
   2. Nechitaylo T
   3. Engl T
   4. Vogel H
   5. Koehler S
   6. Kaltenpoth M

   (2019) Genbank

   ID SAMN11793601. RNA-Seq of Philanthus triangulum: female antenna rRNA depleted, polyA-enriched.

   https://www.ncbi.nlm.nih.gov/biosample/11793601

1. 1. Regulski M
   2. Tully T

   (1995) GenBank

   ID U25117.1. Drosophila melanogaster Ca/calmodulin-dependent nitric oxide synthase (NOS) mRNA, complete cds.

   https://www.ncbi.nlm.nih.gov/nuccore/U25117.1
2. 1. Watanabe T
   2. Shiga T
   3. Yamamoto T
   4. Suzuki N
   5. Ito E

   (2005) GenBank

   ID AB204558.1. Apis mellifera AmNOS mRNA for nitric oxide synthase, complete cds.

   https://www.ncbi.nlm.nih.gov/nuccore/AB204558.1
3. 1. Luckhart S
   2. Vodovotz Y
   3. Cui L

   (1999) GenBank

   ID AH007775.1. Anopheles stephensi nitric oxide synthase gene, complete cds.

   https://www.ncbi.nlm.nih.gov/nuccore/AH007775.1
4. 1. Yuda M

   (1996) GenBank

   ID U59389.1. Rhodnius prolixus salivary gland nitric oxide synthase mRNA, complete cds.

   https://www.ncbi.nlm.nih.gov/nuccore/U59389.1
5. 1. Nighorn A
   2. Gibson NJ
   3. Rivers DM
   4. Hildebrand JG
   5. Morton DB

   (1998) GenBank

   ID AF062749.1. Manduca sexta nitric oxide synthase (NOS) mRNA, complete cds.

   https://www.ncbi.nlm.nih.gov/nuccore/AF062749.1
6. 1. Werren et al

   (2016) GenBank

   ID NM_001168232.1. Nasonia vitripennis nitric oxide synthase (Nos), mRNA.

   https://www.ncbi.nlm.nih.gov/nuccore/NM_001168232.1

1. Report

   1. Administration USOSaH

   (2014) Directorate of Technical Support and Emergency Management (DTSEM)

   United States Department of Labor.

   https://www.osha.gov/dts/

   • Google Scholar
2. 1. Akira S
   2. Uematsu S
   3. Takeuchi O

   (2006) Pathogen recognition and innate immunity

   Cell 124:783–801.

   https://doi.org/10.1016/j.cell.2006.02.015

   • PubMed
   • Google Scholar
3. 1. Akman Gündüz E
   2. Douglas AE

   (2009) Symbiotic bacteria enable insect to use a nutritionally inadequate diet

   Proceedings of the Royal Society B: Biological Sciences 276:987–991.

   https://doi.org/10.1098/rspb.2008.1476

   • Google Scholar
4. 1. Allen MD

   (1959)

   Respiration rates of worker honeybees of different ages and at different temperatures

   Journal of Experimental Biology 36:92–101.

   • Google Scholar
5. 1. Andersen DS
   2. Colombani J
   3. Léopold P

   (2013) Coordination of organ growth: principles and outstanding questions from the world of insects

   Trends in Cell Biology 23:336–344.

   https://doi.org/10.1016/j.tcb.2013.03.005

   • PubMed
   • Google Scholar
6. 1. Arce AN
   2. Smiseth PT
   3. Rozen DE

   (2013) Antimicrobial secretions and social immunity in larval burying beetles, nicrophorus vespilloides

   Animal Behaviour 86:741–745.

   https://doi.org/10.1016/j.anbehav.2013.07.008

   • Google Scholar
7. Book

   1. Atlas RM

   (2004)

   Handbook of Microbiological Media

   CRC press.

   • Google Scholar
8. 1. Barbehenn RV
   2. Reese JC
   3. Hagen KS

   (1999)

   Ecol Entomol

   The Food of Insects, Ecol Entomol, USA, NY-New York, J Wiley & Sons.

   • Google Scholar
9. 1. Barrett M
   2. Schmidt JM

   (1991) A comparison between the amino acid composition of an egg parasitoid wasp and some of its hosts

   Entomologia Experimentalis Et Applicata 59:29–41.

   https://doi.org/10.1111/j.1570-7458.1991.tb01483.x

   • Google Scholar
10. 1. Beckman JS
    2. Koppenol WH

    (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly

    American Journal of Physiology-Cell Physiology 271:C1424–C1437.

    https://doi.org/10.1152/ajpcell.1996.271.5.C1424

    • Google Scholar
11. 1. Berenbaum MR

    (1995) Turnabout is fair play: secondary roles for primary compounds

    Journal of Chemical Ecology 21:925–940.

    https://doi.org/10.1007/BF02033799

    • PubMed
    • Google Scholar
12. 1. Bogdan C
    2. Röllinghoff M
    3. Diefenbach A

    (2000) Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity

    Current Opinion in Immunology 12:64–76.

    https://doi.org/10.1016/S0952-7915(99)00052-7

    • PubMed
    • Google Scholar
13. 1. Bogdan C

    (2001) Nitric oxide and the immune response

    Nature Immunology 2:907–916.

    https://doi.org/10.1038/ni1001-907

    • PubMed
    • Google Scholar
14. 1. Burke AJ
    2. Sullivan FJ
    3. Giles FJ
    4. Glynn SA

    (2013) The yin and yang of nitric oxide in cancer progression

    Carcinogenesis 34:503–512.

    https://doi.org/10.1093/carcin/bgt034

    • PubMed
    • Google Scholar
15. 1. Calabrese V
    2. Mancuso C
    3. Calvani M
    4. Rizzarelli E
    5. Butterfield DA
    6. Stella AM

    (2007) Nitric oxide in the central nervous system: neuroprotection versus neurotoxicity

    Nature Reviews Neuroscience 8:766–775.

    https://doi.org/10.1038/nrn2214

    • PubMed
    • Google Scholar
16. 1. Canessa P
    2. Larrondo LF

    (2013) Environmental responses and the control of iron homeostasis in fungal systems

    Applied Microbiology and Biotechnology 97:939–955.

    https://doi.org/10.1007/s00253-012-4615-x

    • PubMed
    • Google Scholar
17. 1. Castillo L
    2. Beaumier L
    3. Ajami AM
    4. Young VR

    (1996) Whole body nitric oxide synthesis in healthy men determined from [15N] arginine-to-[15N]citrulline labeling

    PNAS 93:11460–11465.

    https://doi.org/10.1073/pnas.93.21.11460

    • PubMed
    • Google Scholar
18. 1. Davies S

    (2000) Nitric oxide signalling in insects

    Insect Biochemistry and Molecular Biology 30:1123–1138.

    https://doi.org/10.1016/S0965-1748(00)00118-1

    • PubMed
    • Google Scholar
19. 1. de Groot AP

    (1952) Amino acid requirements for growth of the honeybee (Apis mellifica L.)

    Experientia 8:192–194.

    https://doi.org/10.1007/BF02173740

    • PubMed
    • Google Scholar
20. 1. Degenkolb T
    2. Düring RA
    3. Vilcinskas A

    (2011) Secondary metabolites released by the burying beetle nicrophorus vespilloides: chemical analyses and possible ecological functions

    Journal of Chemical Ecology 37:724–735.

    https://doi.org/10.1007/s10886-011-9978-4

    • PubMed
    • Google Scholar
21. 1. Engl T
    2. Bodenstein B
    3. Strohm E

    (2016) Mycobiota in the brood cells of the european beewolf, philanthus triangulum (Hymenoptera: crabronidae)

    European Journal of Entomology 113:271–277.

    https://doi.org/10.14411/eje.2016.033

    • Google Scholar
22. 1. Engl T
    2. Kroiss J
    3. Kai M
    4. Nechitaylo TY
    5. Svatoš A
    6. Kaltenpoth M

    (2018) Evolutionary stability of antibiotic protection in a defensive symbiosis

    PNAS 115:E2020–E2029.

    https://doi.org/10.1073/pnas.1719797115

    • PubMed
    • Google Scholar
23. 1. Erhardt A
    2. Rusterholz HP

    (1998) Do peacock butterflies (Inachis io L.) detect and prefer nectar amino acids and other nitrogenous compounds?

    Oecologia 117:536–542.

    https://doi.org/10.1007/s004420050690

    • PubMed
    • Google Scholar
24. 1. Fang FC

    (1997) Perspectives series: host/pathogen interactions. Mechanisms of nitric oxide-related antimicrobial activity

    Journal of Clinical Investigation 99:2818–2825.

    https://doi.org/10.1172/JCI119473

    • PubMed
    • Google Scholar
25. 1. Fang FC

    (2004) Antimicrobial reactive oxygen and nitrogen species: concepts and controversies

    Nature Reviews Microbiology 2:820–832.

    https://doi.org/10.1038/nrmicro1004

    • Google Scholar
26. 1. Feelisch M

    (2008) The chemical biology of nitric oxide--an outsider's reflections about its role in osteoarthritis

    Osteoarthritis and Cartilage 16:S3–S13.

    https://doi.org/10.1016/S1063-4584(08)60007-2

    • PubMed
    • Google Scholar
27. 1. Filipović MR
    2. Koh AC
    3. Arbault S
    4. Niketić V
    5. Debus A
    6. Schleicher U
    7. Bogdan C
    8. Guille M
    9. Lemaître F
    10. Amatore C
    11. Ivanović-Burmazović I

    (2010) Striking inflammation from both sides: manganese(II) pentaazamacrocyclic SOD mimics act also as nitric oxide dismutases: a single-cell study

    Angewandte Chemie International Edition 49:4228–4232.

    https://doi.org/10.1002/anie.200905936

    • PubMed
    • Google Scholar
28. 1. Fisher RC

    (1963)

    Oxygen requirements and the physiological suppression of supernumerary insect parasitoids

    Journal of Experimental Biology 40:531–540.

    • Google Scholar
29. 1. Flórez LV
    2. Biedermann PH
    3. Engl T
    4. Kaltenpoth M

    (2015) Defensive symbioses of animals with prokaryotic and eukaryotic microorganisms

    Natural Product Reports 32:904–936.

    https://doi.org/10.1039/C5NP00010F

    • PubMed
    • Google Scholar
30. 1. Flórez LV
    2. Scherlach K
    3. Gaube P
    4. Ross C
    5. Sitte E
    6. Hermes C
    7. Rodrigues A
    8. Hertweck C
    9. Kaltenpoth M

    (2017) Antibiotic-producing symbionts dynamically transition between plant pathogenicity and insect-defensive mutualism

    Nature Communications 8:15172.

    https://doi.org/10.1038/ncomms15172

    • PubMed
    • Google Scholar
31. 1. Förstermann U
    2. Sessa WC

    (2012) Nitric oxide synthases: regulation and function

    European Heart Journal 33:829–837.

    https://doi.org/10.1093/eurheartj/ehr304

    • PubMed
    • Google Scholar
32. 1. Gao SH
    2. Fan L
    3. Yuan Z
    4. Bond PL

    (2015) The concentration-determined and population-specific antimicrobial effects of free nitrous acid on pseudomonas aeruginosa PAO1

    Applied Microbiology and Biotechnology 99:2305–2312.

    https://doi.org/10.1007/s00253-014-6211-8

    • PubMed
    • Google Scholar
33. 1. Ghaffari A
    2. Miller CC
    3. McMullin B
    4. Ghahary A

    (2006) Potential application of gaseous nitric oxide as a topical antimicrobial agent

    Nitric Oxide 14:21–29.

    https://doi.org/10.1016/j.niox.2005.08.003

    • PubMed
    • Google Scholar
34. 1. Gilliam M
    2. TABER S
    3. Richardson GV

    (1983) Hygienic behavior of honey bees in relation to chalkbrood disease

    Apidologie 14:29–39.

    https://doi.org/10.1051/apido:19830103

    • Google Scholar
35. 1. Gorman MJ
    2. Kankanala P
    3. Kanost MR

    (2004) Bacterial challenge stimulates innate immune responses in extra-embryonic tissues of tobacco hornworm eggs

    Insect Molecular Biology 13:19–24.

    https://doi.org/10.1111/j.1365-2583.2004.00454.x

    • PubMed
    • Google Scholar
36. 1. Gross J
    2. Schumacher K
    3. Schmidtberg H
    4. Vilcinskas A

    (2008) Protected by fumigants: beetle perfumes in antimicrobial defense

    Journal of Chemical Ecology 34:179–188.

    https://doi.org/10.1007/s10886-007-9416-9

    • PubMed
    • Google Scholar
37. 1. Gross J
    2. Schmidtberg H

    (2009)

    Research on Chrysomelidae

    177–190, Glands of leaf beetle larvae–protective structures against attacking predators and pathogens, Research on Chrysomelidae, Brill.

    • Google Scholar
38. 1. Guevara I
    2. Iwanejko J
    3. Dembińska-Kieć A
    4. Pankiewicz J
    5. Wanat A
    6. Anna P
    7. Gołąbek I
    8. Bartuś S
    9. Malczewska-Malec M
    10. Szczudlik A

    (1998) Determination of nitrite/nitrate in human biological material by the simple griess reaction

    Clinica Chimica Acta 274:177–188.

    https://doi.org/10.1016/S0009-8981(98)00060-6

    • Google Scholar
39. 1. Guzik TJ
    2. Korbut R
    3. Adamek-Guzik T

    (2003)

    Nitric oxide and superoxide in inflammation and immune regulation

    Journal of Physiology and Pharmacology 54:469–487.

    • PubMed
    • Google Scholar
40. Software

    1. Hammer Ø
    2. Harper D
    3. Ryan P

    (2001)

    PAST-Palaeontological statistics

    Palaeontological Association.
41. 1. Hazen SL
    2. Hsu FF
    3. Mueller DM
    4. Crowley JR
    5. Heinecke JW

    (1996) Human neutrophils employ chlorine gas as an oxidant during phagocytosis

    Journal of Clinical Investigation 98:1283–1289.

    https://doi.org/10.1172/JCI118914

    • PubMed
    • Google Scholar
42. 1. Herzner G
    2. Schmitt T
    3. Peschke K
    4. Hilpert A
    5. Strohm E

    (2007) Food wrapping with the postpharyngeal gland secretion by females of the european beewolf philanthus triangulum

    Journal of Chemical Ecology 33:849–859.

    https://doi.org/10.1007/s10886-007-9263-8

    • PubMed
    • Google Scholar
43. 1. Herzner G
    2. Ruther J
    3. Goller S
    4. Schulz S
    5. Goettler W
    6. Strohm E

    (2011a) Structure, chemical composition and putative function of the postpharyngeal gland of the emerald cockroach wasp, ampulex compressa (Hymenoptera, ampulicidae)

    Zoology 114:36–45.

    https://doi.org/10.1016/j.zool.2010.10.002

    • PubMed
    • Google Scholar
44. 1. Herzner G
    2. Engl T
    3. Strohm E

    (2011b) Cryptic combat against competing microbes is a costly component of parental care in a digger wasp

    Animal Behaviour 82:321–328.

    https://doi.org/10.1016/j.anbehav.2011.05.006

    • Google Scholar
45. 1. Herzner G
    2. Schlecht A
    3. Dollhofer V
    4. Parzefall C
    5. Harrar K
    6. Kreuzer A
    7. Pilsl L
    8. Ruther J

    (2013) Larvae of the parasitoid wasp ampulex compressa sanitize their host, the american cockroach, with a blend of antimicrobials

    PNAS 110:1369–1374.

    https://doi.org/10.1073/pnas.1213384110

    • PubMed
    • Google Scholar
46. 1. Herzner G
    2. Strohm E

    (2007) Fighting fungi with physics: food wrapping by a solitary wasp prevents water condensation

    Current Biology 17:R46–R47.

    https://doi.org/10.1016/j.cub.2006.11.060

    • PubMed
    • Google Scholar
47. 1. Herzner G
    2. Strohm E

    (2008) Food wrapping by females of the european beewolf, philanthus triangulum, retards water loss of larval provisions

    Physiological Entomology 33:101–109.

    https://doi.org/10.1111/j.1365-3032.2007.00603.x

    • Google Scholar
48. Book

    1. Hirano M
    2. Das S
    3. Guo P
    4. Cooper MD

    (2011)

    The Evolution of Adaptive Immunity in Vertebrates

    In: Alt F. W, Austen K. F, Honjo T, Melchers F, Uhr J. W, Unanue E. R, editors. Advances in Immunology, 109. San Diego: Elsevier Academic Press Inc. pp. 125–157.

    • Google Scholar
49. 1. Holm S

    (1979)

    A simple sequentially rejective multiple test procedure 

    Scandinavian Journal of Statistics 6:65–70.

    • Google Scholar
50. 1. Hooper LV
    2. Littman DR
    3. Macpherson AJ

    (2012) Interactions between the microbiota and the immune system

    Science 336:1268–1273.

    https://doi.org/10.1126/science.1223490

    • PubMed
    • Google Scholar
51. 1. Huelsenbeck JP
    2. Ronquist F
    3. Nielsen R
    4. Bollback JP

    (2001) Bayesian inference of phylogeny and its impact on evolutionary biology

    Science 294:2310–2314.

    https://doi.org/10.1126/science.1065889

    • PubMed
    • Google Scholar
52. 1. Huelsenbeck JP
    2. Ronquist F

    (2001) MRBAYES: Bayesian inference of phylogenetic trees

    Bioinformatics 17:754–755.

    https://doi.org/10.1093/bioinformatics/17.8.754

    • PubMed
    • Google Scholar
53. 1. Iwasaki A
    2. Medzhitov R

    (2010) Regulation of adaptive immunity by the innate immune system

    Science 327:291–295.

    https://doi.org/10.1126/science.1183021

    • PubMed
    • Google Scholar
54. Book

    1. Jander G
    2. Blasius E

    (1971)

    Einführung in Das Anorganisch-Chemische Praktikum

    Stuttgart: Hirzel.

    • Google Scholar
55. 1. Janzen DH

    (1977) Why fruits rot, seeds mold, and meat spoils

    The American Naturalist 111:691–713.

    https://doi.org/10.1086/283200

    • Google Scholar
56. 1. Jones ML
    2. Ganopolsky JG
    3. Labbé A
    4. Wahl C
    5. Prakash S

    (2010) Antimicrobial properties of nitric oxide and its application in antimicrobial formulations and medical devices

    Applied Microbiology and Biotechnology 88:401–407.

    https://doi.org/10.1007/s00253-010-2733-x

    • PubMed
    • Google Scholar
57. 1. Kaltenpoth M
    2. Göttler W
    3. Herzner G
    4. Strohm E

    (2005) Symbiotic bacteria protect wasp larvae from fungal infestation

    Current Biology 15:475–479.

    https://doi.org/10.1016/j.cub.2004.12.084

    • PubMed
    • Google Scholar
58. 1. Kaltenpoth M
    2. Schmitt T
    3. Strohm E

    (2009) Hydrocarbons in the antennal gland secretion of female european beewolves, philanthus triangulum (Hymenoptera, crabronidae)

    Chemoecology 19:219–225.

    https://doi.org/10.1007/s00049-009-0022-x

    • Google Scholar
59. 1. Kaltenpoth M
    2. Roeser-Mueller K
    3. Koehler S
    4. Peterson A
    5. Nechitaylo TY
    6. Stubblefield JW
    7. Herzner G
    8. Seger J
    9. Strohm E

    (2014) Partner choice and fidelity stabilize coevolution in a Cretaceous-age defensive symbiosis

    PNAS 111:6359–6364.

    https://doi.org/10.1073/pnas.1400457111

    • PubMed
    • Google Scholar
60. 1. Kojima H
    2. Hirotani M
    3. Urano Y
    4. Kikuchi K
    5. Higuchi T
    6. Nagano T

    (2000) Fluorescent indicators for nitric oxide based on rhodamine chromophore

    Tetrahedron Letters 41:69–72.

    https://doi.org/10.1016/S0040-4039(99)02002-X

    • Google Scholar
61. 1. Kroiss J
    2. Schmitt T
    3. Strohm E

    (2009) Low level of cuticular hydrocarbons in a parasitoid of a solitary digger wasp and its potential for concealment

    Entomological Science 12:9–16.

    https://doi.org/10.1111/j.1479-8298.2009.00300.x

    • Google Scholar
62. 1. Kroiss J
    2. Kaltenpoth M
    3. Schneider B
    4. Schwinger MG
    5. Hertweck C
    6. Maddula RK
    7. Strohm E
    8. Svatos A

    (2010) Symbiotic streptomycetes provide antibiotic combination prophylaxis for wasp offspring

    Nature Chemical Biology 6:261–263.

    https://doi.org/10.1038/nchembio.331

    • PubMed
    • Google Scholar
63. 1. Labbé P
    2. McTaggart SJ
    3. Little TJ

    (2009) An ancient immunity gene duplication in Daphnia magna: rna expression and sequence analysis of two nitric oxide synthase genes

    Developmental & Comparative Immunology 33:1000–1010.

    https://doi.org/10.1016/j.dci.2009.04.006

    • PubMed
    • Google Scholar
64. 1. Lai T
    2. Li B
    3. Qin G
    4. Tian S

    (2011) Oxidative damage involves in the inhibitory effect of nitric oxide on spore germination of penicillium expansum

    Current Microbiology 62:229–234.

    https://doi.org/10.1007/s00284-010-9695-1

    • PubMed
    • Google Scholar
65. 1. Lazar EE
    2. Wills RB
    3. Ho BT
    4. Harris AM
    5. Spohr LJ

    (2008) Antifungal effect of gaseous nitric oxide on mycelium growth, sporulation and spore germination of the postharvest horticulture pathogens, Aspergillus Niger, monilinia fructicola and penicillium italicum

    Letters in Applied Microbiology 46:688–692.

    https://doi.org/10.1111/j.1472-765X.2008.02373.x

    • PubMed
    • Google Scholar
66. 1. Lemaitre B
    2. Hoffmann J

    (2007) The host defense of Drosophila melanogaster

    Annual Review of Immunology 25:697–743.

    https://doi.org/10.1146/annurev.immunol.25.022106.141615

    • PubMed
    • Google Scholar
67. Book

    1. Lide DR

    (1995)

    CRC Handbook of Chemistry and Physics: A Ready-Reference Book of Chemical and Physical Data

    CRC press.

    • Google Scholar
68. 1. Lopes RB
    2. Laumann RA
    3. Blassioli-Moraes MC
    4. Borges M
    5. Faria M

    (2015) The fungistatic and fungicidal effects of volatiles from metathoracic glands of soybean-attacking stink bugs (Heteroptera: pentatomidae) on the entomopathogen beauveria bassiana

    Journal of Invertebrate Pathology 132:77–85.

    https://doi.org/10.1016/j.jip.2015.08.011

    • PubMed
    • Google Scholar
69. 1. Luckhart S
    2. Vodovotz Y
    3. Cui L
    4. Rosenberg R

    (1998) The mosquito anopheles stephensi limits malaria parasite development with inducible synthesis of nitric oxide

    PNAS 95:5700–5705.

    https://doi.org/10.1073/pnas.95.10.5700

    • PubMed
    • Google Scholar
70. 1. Luckhart S
    2. Li K

    (2001) Transcriptional complexity of the anopheles stephensi nitric oxide synthase gene

    Insect Biochemistry and Molecular Biology 31:249–256.

    https://doi.org/10.1016/S0965-1748(00)00144-2

    • PubMed
    • Google Scholar
71. 1. Lüth HJ
    2. Münch G
    3. Arendt T

    (2002) Aberrant expression of NOS isoforms in Alzheimer's disease is structurally related to nitrotyrosine formation

    Brain Research 953:135–143.

    https://doi.org/10.1016/S0006-8993(02)03280-8

    • PubMed
    • Google Scholar
72. 1. Marchini D
    2. Marri L
    3. Rosetto M
    4. Manetti AG
    5. Dallai R

    (1997) Presence of antibacterial peptides on the laid egg chorion of the medfly ceratitis capitata

    Biochemical and Biophysical Research Communications 240:657–663.

    https://doi.org/10.1006/bbrc.1997.7694

    • PubMed
    • Google Scholar
73. 1. Martins VP
    2. Dinamarco TM
    3. Curti C
    4. Uyemura SA

    (2011) Classical and alternative components of the mitochondrial respiratory chain in pathogenic fungi as potential therapeutic targets

    Journal of Bioenergetics and Biomembranes 43:81–88.

    https://doi.org/10.1007/s10863-011-9331-1

    • PubMed
    • Google Scholar
74. 1. Mesquita RD
    2. Vionette-Amaral RJ
    3. Lowenberger C
    4. Rivera-Pomar R
    5. Monteiro FA
    6. Minx P
    7. Spieth J
    8. Carvalho AB
    9. Panzera F
    10. Lawson D
    11. Torres AQ
    12. Ribeiro JM
    13. Sorgine MH
    14. Waterhouse RM
    15. Montague MJ
    16. Abad-Franch F
    17. Alves-Bezerra M
    18. Amaral LR
    19. Araujo HM
    20. Araujo RN
    21. Aravind L
    22. Atella GC
    23. Azambuja P
    24. Berni M
    25. Bittencourt-Cunha PR
    26. Braz GR
    27. Calderón-Fernández G
    28. Carareto CM
    29. Christensen MB
    30. Costa IR
    31. Costa SG
    32. Dansa M
    33. Daumas-Filho CR
    34. De-Paula IF
    35. Dias FA
    36. Dimopoulos G
    37. Emrich SJ
    38. Esponda-Behrens N
    39. Fampa P
    40. Fernandez-Medina RD
    41. da Fonseca RN
    42. Fontenele M
    43. Fronick C
    44. Fulton LA
    45. Gandara AC
    46. Garcia ES
    47. Genta FA
    48. Giraldo-Calderón GI
    49. Gomes B
    50. Gondim KC
    51. Granzotto A
    52. Guarneri AA
    53. Guigó R
    54. Harry M
    55. Hughes DS
    56. Jablonka W
    57. Jacquin-Joly E
    58. Juárez MP
    59. Koerich LB
    60. Lange AB
    61. Latorre-Estivalis JM
    62. Lavore A
    63. Lawrence GG
    64. Lazoski C
    65. Lazzari CR
    66. Lopes RR
    67. Lorenzo MG
    68. Lugon MD
    69. Majerowicz D
    70. Marcet PL
    71. Mariotti M
    72. Masuda H
    73. Megy K
    74. Melo AC
    75. Missirlis F
    76. Mota T
    77. Noriega FG
    78. Nouzova M
    79. Nunes RD
    80. Oliveira RL
    81. Oliveira-Silveira G
    82. Ons S
    83. Orchard I
    84. Pagola L
    85. Paiva-Silva GO
    86. Pascual A
    87. Pavan MG
    88. Pedrini N
    89. Peixoto AA
    90. Pereira MH
    91. Pike A
    92. Polycarpo C
    93. Prosdocimi F
    94. Ribeiro-Rodrigues R
    95. Robertson HM
    96. Salerno AP
    97. Salmon D
    98. Santesmasses D
    99. Schama R
    100. Seabra-Junior ES
    101. Silva-Cardoso L
    102. Silva-Neto MA
    103. Souza-Gomes M
    104. Sterkel M
    105. Taracena ML
    106. Tojo M
    107. Tu ZJ
    108. Tubio JM
    109. Ursic-Bedoya R
    110. Venancio TM
    111. Walter-Nuno AB
    112. Wilson D
    113. Warren WC
    114. Wilson RK
    115. Huebner E
    116. Dotson EM
    117. Oliveira PL

    (2015) Genome of Rhodnius Prolixus, an insect vector of chagas disease, reveals unique adaptations to hematophagy and parasite infection

    PNAS 112:14936–14941.

    https://doi.org/10.1073/pnas.1506226112

    • PubMed
    • Google Scholar
75. 1. Moroz LL
    2. Kohn AB

    (2007) On the comparative biology of nitric oxide (NO) synthetic pathways: parallel evolution of NO-mediated signaling

    Advances in Experimental Biology 1:1–44.

    https://doi.org/10.1016/S1872-2423(07)01001-0

    • Google Scholar
76. 1. Morris Jr SM

    (1999)

    Arginine synthesis, metabolism, and transport: regulators of nitric oxide synthesis

    Cellular and Molecular Biology of Nitric Oxide pp. 57–85.

    • Google Scholar
77. 1. Morris Jr SM

    (2000)

    Regulation of arginine availability and its impact on NO synthesis

    Nitric Oxide: Biology and Pathobiology pp. 187–197.

    • Google Scholar
78. Book

    1. Mücke W
    2. Lemmen C

    (2010)

    Duft Und Und Geruch: Wirkungen Und Gesundheitliche Bedeutung Von Geruchsstoffen

    Landsberg am Lech, Germany: ecomed-Storck GmbH.

    • Google Scholar
79. 1. Müller U

    (1994)

    Ca2+/calmodulin-dependent nitric oxide synthase in apis mellifera and Drosophila melanogaster

    The European Journal of Neuroscience 6:1362–1370.

    • PubMed
    • Google Scholar
80. 1. Mur LA
    2. Mandon J
    3. Cristescu SM
    4. Harren FJ
    5. Prats E

    (2011) Methods of nitric oxide detection in plants: a commentary

    Plant Science 181:509–519.

    https://doi.org/10.1016/j.plantsci.2011.04.003

    • PubMed
    • Google Scholar
81. 1. O'Brien DM
    2. Boggs CL
    3. Fogel ML

    (2003) Pollen feeding in the butterfly Heliconius charitonia : isotopic evidence for essential amino acid transfer from pollen to eggs

    Proceedings of the Royal Society of London. Series B: Biological Sciences 270:2631–2636.

    https://doi.org/10.1098/rspb.2003.2552

    • Google Scholar
82. 1. Otti O
    2. Tragust S
    3. Feldhaar H

    (2014) Unifying external and internal immune defences

    Trends in Ecology & Evolution 29:625–634.

    https://doi.org/10.1016/j.tree.2014.09.002

    • PubMed
    • Google Scholar
83. 1. Pacher P
    2. Beckman JS
    3. Liaudet L

    (2007) Nitric oxide and peroxynitrite in health and disease

    Physiological Reviews 87:315–424.

    https://doi.org/10.1152/physrev.00029.2006

    • Google Scholar
84. 1. Pautz A
    2. Art J
    3. Hahn S
    4. Nowag S
    5. Voss C
    6. Kleinert H

    (2010) Regulation of the expression of inducible nitric oxide synthase

    Nitric Oxide 23:75–93.

    https://doi.org/10.1016/j.niox.2010.04.007

    • PubMed
    • Google Scholar
85. 1. Payne SH
    2. Loomis WF

    (2006) Retention and loss of amino acid biosynthetic pathways based on analysis of whole-genome sequences

    Eukaryotic Cell 5:272–276.

    https://doi.org/10.1128/EC.5.2.272-276.2006

    • PubMed
    • Google Scholar
86. 1. Peterson DA
    2. Peterson DC
    3. Archer S
    4. Weir EK

    (1992) The non specificity of specific nitric oxide synthase inhibitors

    Biochemical and Biophysical Research Communications 187:797–801.

    https://doi.org/10.1016/0006-291X(92)91266-S

    • PubMed
    • Google Scholar
87. Book

    1. Poole R

    (2005)

    Nitric Oxide and Nitrosative Stress Tolerance in Bacteria

    Portland Press Limited.

    • Google Scholar
88. 1. Price MN
    2. Dehal PS
    3. Arkin AP

    (2009) FastTree: computing large minimum evolution trees with profiles instead of a distance matrix

    Molecular Biology and Evolution 26:1641–1650.

    https://doi.org/10.1093/molbev/msp077

    • PubMed
    • Google Scholar
89. 1. Price MN
    2. Dehal PS
    3. Arkin AP

    (2010) FastTree 2--approximately maximum-likelihood trees for large alignments

    PLOS ONE 5:e9490.

    https://doi.org/10.1371/journal.pone.0009490

    • PubMed
    • Google Scholar
90. 1. Regulski M
    2. Tully T

    (1995) Molecular and biochemical characterization of dNOS: a Drosophila Ca2+/calmodulin-dependent nitric oxide synthase

    PNAS 92:9072–9076.

    https://doi.org/10.1073/pnas.92.20.9072

    • PubMed
    • Google Scholar
91. 1. Rivero A

    (2006) Nitric oxide: an antiparasitic molecule of invertebrates

    Trends in Parasitology 22:219–225.

    https://doi.org/10.1016/j.pt.2006.02.014

    • PubMed
    • Google Scholar
92. 1. Ronquist F
    2. Huelsenbeck JP

    (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models

    Bioinformatics 19:1572–1574.

    https://doi.org/10.1093/bioinformatics/btg180

    • PubMed
    • Google Scholar
93. Book

    1. Röszer T

    (2012)

    The Biology of Subcellular Nitric Oxide

    Heidleberg: Springer.

    • Google Scholar
94. 1. Rozen DE
    2. Engelmoer DJ
    3. Smiseth PT

    (2008) Antimicrobial strategies in burying beetles breeding on carrion

    PNAS 105:17890–17895.

    https://doi.org/10.1073/pnas.0805403105

    • PubMed
    • Google Scholar
95. 1. Salerno JC
    2. Harris DE
    3. Irizarry K
    4. Patel B
    5. Morales AJ
    6. Smith SM
    7. Martasek P
    8. Roman LJ
    9. Masters BS
    10. Jones CL
    11. Weissman BA
    12. Lane P
    13. Liu Q
    14. Gross SS

    (1997) An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase

    Journal of Biological Chemistry 272:29769–29777.

    https://doi.org/10.1074/jbc.272.47.29769

    • PubMed
    • Google Scholar
96. Book

    1. Sambrook J
    2. Russell DW

    (2001a)

    Molecular Cloning: A Laboratory Manual, 1-3

    Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

    • Google Scholar
97. Book

    1. Sambrook J
    2. Russell DW

    (2001b)

    Molecular Cloning: A Laboratory Manual, 3

    Cold Spring Harbor Laboratory Press.

    • Google Scholar
98. 1. Sasaki T
    2. Ishikawa H

    (1995) Production of essential amino acids from glutamate by mycetocyte symbionts of the pea aphid, acyrthosiphon pisum

    Journal of Insect Physiology 41:41–46.

    https://doi.org/10.1016/0022-1910(94)00080-Z

    • Google Scholar
99. 1. Shukla SP
    2. Plata C
    3. Reichelt M
    4. Steiger S
    5. Heckel DG
    6. Kaltenpoth M
    7. Vilcinskas A
    8. Vogel H

    (2018) Microbiome-assisted carrion preservation aids larval development in a burying beetle

    PNAS 115:11274–11279.

    https://doi.org/10.1073/pnas.1812808115

    • PubMed
    • Google Scholar
100. Book

     1. Siva-Jothy MT
     2. Moret Y
     3. Rolff J

     (2005)

     Insect immunity: An evolutionary ecology perspective

     In: Simpson S. J, editors. Advances in Insect Physiology, 32. San Diego: Elsevier Academic Press Inc. pp. 1–48.

     • Google Scholar
101. 1. Smith BC
     2. Underbakke ES
     3. Kulp DW
     4. Schief WR
     5. Marletta MA

     (2013) Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation

     PNAS 110:E3577–E3586.

     https://doi.org/10.1073/pnas.1313331110

     • PubMed
     • Google Scholar
102. 1. Soegiarto L
     2. Wills RBH
     3. Seberry JA
     4. Leshem YY

     (2003) Nitric oxide degradation in oxygen atmospheres and rate of uptake by horticultural produce

     Postharvest Biology and Technology 28:327–331.

     https://doi.org/10.1016/S0925-5214(02)00199-0

     • Google Scholar
103. 1. Strohm E

     (2000) Factors affecting body size and fat content in a digger wasp

     Oecologia 123:184–191.

     https://doi.org/10.1007/s004420051004

     • PubMed
     • Google Scholar
104. 1. Strohm E
     2. Linsenmair KE

     (1994)

     Leaving the cradle: how beewolves (Philanthus triangulum F.) obtain the necessary spatial information for emergence

     Zoology 95:137–146.

     • Google Scholar
105. 1. Strohm E
     2. Linsenmair KE

     (2000) Allocation of parental investment among individual offspring in the European beewolf Philanthus triangulum F. (Hymenoptera: sphecidae)

     Biological Journal of the Linnean Society 69:173–192.

     https://doi.org/10.1111/j.1095-8312.2000.tb01197.x

     • Google Scholar
106. 1. Strohm E
     2. Linsenmair KE

     (2001) Females of the european beewolf preserve their honeybee prey against competing fungi

     Ecological Entomology 26:198–203.

     https://doi.org/10.1046/j.1365-2311.2001.00300.x

     • Google Scholar
107. 1. Thomas DD
     2. Miranda KM
     3. Citrin D
     4. Espey MG
     5. Wink DA

     (2003)

     Combat Medicine

     23–60, Nitric oxide, Combat Medicine, Springer.

     • Google Scholar
108. 1. Thompson SN

     (1976) The amino acid requirements for larval development of the hymenopterous parasitoid exeristes roborator fabricius (hymenoptera: ichneumonidae)

     Comparative Biochemistry and Physiology Part A: Physiology 53:211–213.

     https://doi.org/10.1016/S0300-9629(76)80057-6

     • Google Scholar
109. 1. Titheradge MA

     (1999) Nitric oxide in septic shock

     Biochimica Et Biophysica Acta (BBA) - Bioenergetics 1411:437–455.

     https://doi.org/10.1016/S0005-2728(99)00031-6

     • Google Scholar
110. 1. Tragust S
     2. Mitteregger B
     3. Barone V
     4. Konrad M
     5. Ugelvig LV
     6. Cremer S

     (2013) Ants disinfect fungus-exposed brood by oral uptake and spread of their poison

     Current Biology 23:76–82.

     https://doi.org/10.1016/j.cub.2012.11.034

     • PubMed
     • Google Scholar
111. 1. Tranter C
     2. Graystock P
     3. Shaw C
     4. Lopes JFS
     5. Hughes WOH

     (2014) Sanitizing the fortress: protection of ant brood and nest material by worker antibiotics

     Behavioral Ecology and Sociobiology 68:499–507.

     https://doi.org/10.1007/s00265-013-1664-9

     • Google Scholar
112. 1. Tsikas D

     (2007) Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: appraisal of the Griess reaction in the l-arginine/nitric oxide area of research

     Journal of Chromatography B 851:51–70.

     https://doi.org/10.1016/j.jchromb.2006.07.054

     • Google Scholar
113. 1. Uchida K

     (1993) Balanced amino acid composition essential for infusion-induced egg development in the mosquito (Culex pipiens pallens)

     Journal of Insect Physiology 39:615–621.

     https://doi.org/10.1016/0022-1910(93)90044-R

     • Google Scholar
114. 1. Vander Meer RK
     2. Morel L

     (1995) Ant queens deposit pheromones and antimicrobial agents on eggs

     Naturwissenschaften 82:93–95.

     https://doi.org/10.1007/BF01140150

     • Google Scholar
115. 1. Vaughn MW
     2. Kuo L
     3. Liao JC

     (1998) Estimation of nitric oxide production and reactionrates in tissue by use of a mathematical model

     American Journal of Physiology-Heart and Circulatory Physiology 274:H2163–H2176.

     https://doi.org/10.1152/ajpheart.1998.274.6.H2163

     • Google Scholar
116. 1. Vilcinskas A
     2. Mukherjee K
     3. Vogel H

     (2013) Expansion of the antimicrobial peptide repertoire in the invasive ladybird Harmonia axyridis

     Proceedings of the Royal Society B: Biological Sciences 280:20122113.

     https://doi.org/10.1098/rspb.2012.2113

     • Google Scholar
117. 1. Virgili M
     2. Poli A
     3. Beraudi A
     4. Giuliani A
     5. Villani L

     (2001) Regional distribution of nitric oxide synthase and NADPH-diaphorase activities in the central nervous system of teleosts

     Brain Research 901:202–207.

     https://doi.org/10.1016/S0006-8993(01)02357-5

     • PubMed
     • Google Scholar
118. 1. Wang J
     2. Higgins VJ

     (2005) Nitric oxide has a regulatory effect in the germination of conidia of colletotrichum coccodes

     Fungal Genetics and Biology 42:284–292.

     https://doi.org/10.1016/j.fgb.2004.12.006

     • PubMed
     • Google Scholar
119. 1. Wareham LK
     2. Southam HM
     3. Poole RK

     (2018) Do nitric oxide, carbon monoxide and hydrogen sulfide really qualify as 'gasotransmitters' in bacteria?

     Biochemical Society Transactions 46:1107–1118.

     https://doi.org/10.1042/BST20170311

     • PubMed
     • Google Scholar
120. 1. Weiner CN
     2. Hilpert A
     3. Werner M
     4. Linsenmair KE
     5. Blüthgen N

     (2010) Pollen amino acids and flower specialisation in solitary bees

     Apidologie 41:476–487.

     https://doi.org/10.1051/apido/2009083

     • Google Scholar
121. 1. Weiss K
     2. Parzefall C
     3. Herzner G

     (2014) Multifaceted defense against antagonistic microbes in developing offspring of the parasitoid wasp ampulex compressa (Hymenoptera, ampulicidae)

     PLOS ONE 9:e98784.

     https://doi.org/10.1371/journal.pone.0098784

     • PubMed
     • Google Scholar
122. 1. Whitten M
     2. Sun F
     3. Tew I
     4. Schaub G
     5. Soukou C
     6. Nappi A
     7. Ratcliffe N

     (2007) Differential modulation of rhodnius prolixus nitric oxide activities following challenge with trypanosoma rangeli, T. Cruzi and bacterial cell wall components

     Insect Biochemistry and Molecular Biology 37:440–452.

     https://doi.org/10.1016/j.ibmb.2007.02.001

     • PubMed
     • Google Scholar
123. 1. Willmot M
     2. Gibson C
     3. Gray L
     4. Murphy S
     5. Bath P

     (2005) Nitric oxide synthase inhibitors in experimental ischemic stroke and their effects on infarct size and cerebral blood flow: a systematic review

     Free Radical Biology and Medicine 39:412–425.

     https://doi.org/10.1016/j.freeradbiomed.2005.03.028

     • Google Scholar
124. 1. Wilson K
     2. Cotter SC

     (2013) Host-parasite interactions and the evolution of immune defense

     Advances in the Study of Behavior 45:81–174.

     https://doi.org/10.1016/B978-0-12-407186-5.00003-3

     • Google Scholar
125. 1. Wink DA
     2. Hines HB
     3. Cheng RY
     4. Switzer CH
     5. Flores-Santana W
     6. Vitek MP
     7. Ridnour LA
     8. Colton CA

     (2011) Nitric oxide and redox mechanisms in the immune response

     Journal of Leukocyte Biology 89:873–891.

     https://doi.org/10.1189/jlb.1010550

     • PubMed
     • Google Scholar
126. 1. Wong M-L
     2. Rettori V
     3. AL-Shekhlee A
     4. Bongiorno PB
     5. Canteros G
     6. McCann SM
     7. Gold PW
     8. Licinio J

     (1996) Inducible nitric oxide synthase gene expression in the brain during systemic inflammation

     Nature Medicine 2:581–584.

     https://doi.org/10.1038/nm0596-581

     • Google Scholar
127. 1. Wu G
     2. Flynn NE
     3. Flynn SP
     4. Jolly CA
     5. Davis PK

     (1999) Dietary protein or arginine deficiency impairs constitutive and inducible nitric oxide synthesis by young rats

     The Journal of Nutrition 129:1347–1354.

     https://doi.org/10.1093/jn/129.7.1347

     • PubMed
     • Google Scholar
128. 1. Zeidler D
     2. Zähringer U
     3. Gerber I
     4. Dubery I
     5. Hartung T
     6. Bors W
     7. Hutzler P
     8. Durner J

     (2004) Innate immunity in arabidopsis thaliana: lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes

     PNAS 101:15811–15816.

     https://doi.org/10.1073/pnas.0404536101

     • PubMed
     • Google Scholar
129. 1. Zhou S
     2. Fushinobu S
     3. Nakanishi Y
     4. Kim SW
     5. Wakagi T
     6. Shoun H

     (2009) Cloning and characterization of two flavohemoglobins from aspergillus oryzae

     Biochemical and Biophysical Research Communications 381:7–11.

     https://doi.org/10.1016/j.bbrc.2009.01.112

     • PubMed
     • Google Scholar
130. 1. Zhukovskaya M
     2. Yanagawa A
     3. Forschler BT

     (2013) Grooming behavior as a mechanism of insect disease defense

     Insects 4:609–630.

     https://doi.org/10.3390/insects4040609

     • PubMed
     • Google Scholar

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

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