Antimicrobial Defense Strategies in Two Solitary Species 1

Entomologie heute 26 (2014): 1-17

Antimicrobial Defense Strategies in Two Solitary Wasp Species

Strategien antimikrobieller Verteidigung bei zwei solitären Wespen

GUDRUN HERZNER

Summary: Microorganisms are ubiquitous and may act as pathogens as well as food competitors that continuously challenge the survival and well-being of all higher organisms. Owing to their various specifi c life-styles may be exposed to severe and diverse microbial hazards and have consequently evolved an impressive array of sophisticated antimicrobial defenses. In this paper some strategies of antimicrobial defense in two Hymenopteran model systems, the European - wolf Philanthus triangulum (, ) and the Emerald wasp compressa (Hymenoptera, ) will be reviewed. Female P. triangulum provide their offspring with paralyzed honeybees (Apis mellifera) in subterranean nests, where the microclimatic conditions promote growth of mold fungi. To mitigate the risks of fungal infestations females embalm their prey with a secretion of unsaturated hydrocarbons that form an oily layer on the surface of the bees and reduce water condensation from the surrounding air. The induced alteration in microclimatic conditions signifi cantly reduces growth of mold fungi on the provisioned bees and thus enhances survival of the wasp offspring. This benefi t of prey embalming is not to be had for nothing, however, as it reduces the future reproductive ability of the mother. Larvae of A. compressa develop in like the americana. In order to ward off detrimental microbes that are naturally present on the cockroaches, A. compressa larvae sanitize their host cockroaches with large amounts of an antimicrobial secretion. This specifi c cocktail of substances has broad-spectrum activity against a wide range of microbes. Furthermore, the larvae implement the antimicrobials in a spatially and temporally coordinated manner in solution as well as in vapor form. This strategy provides them with a reliable long-term protection via three lines of defense against microbes invading from the nest environment. The examples given here clearly illustrate the ingenuity and diversity of nature’s solutions to microbial hazards. While in the mothers make use of a physical effect to retard growth of mold fungi on larval provisions, in the the larvae themselves fi ght detrimental microbes by deploying a blend of antimicrobial chemicals. Further research on the as yet only scarcely investigated plethora of antimicrobial strategies of insects may inspire new approaches to microbial control in human food industry and medicine to counteract the proliferation of antimicrobial resistant microorganisms.

Keywords: Antimicrobial, antifungal, antibiotic, mold fungi, fumigation

Zusammenfassung: Mikroorganismen sind allgegenwärtig und können sowohl als Pathogene als auch als Nahrungskonkurrenten das Leben aller höheren Organismen gefährden. Aufgrund ihres großen Spektrums unterschiedlicher Lebensweisen können Insekten einer Vielzahl ernster Gefahren durch Mikroorganismen ausgesetzt sein. Folglich haben sie im Laufe der Evolution eine beeindruckende Vielfalt an ausgefeilten antimikrobiellen Verteidigungsstrategien entwickelt. In diesem Artikel werden am Beispiel zweier Modellorganismen, des Europäischen Bienenwolfs Phil- anthus triangulum (Hymenoptera, Crabronidae) und der Juwelwespe Ampulex compressa (Hymenoptera, Ampulicidae), einige antimikrobielle Abwehrstrategien beschrieben. Bienenwolf-Weibchen stellen ihren Nachkommen gelähmte Honigbienen (Apis mellifera) als Nahrung in unterirdischen Nestern zur Verfügung, deren mikroklimatische Bedingungen das Wachstum von Schimmelpilzen stark

Entomologie heute 26 (2014) 2 GUDRUN HERZNER fördern. Um das Verschimmeln des Larvenfutters zu verzögern und zu vermindern, balsamieren die Weibchen ihre Beutebienen mit ungesättigten Kohlenwasserstoffen ein, die auf der Bienen- oberfl äche eine ölige Schicht bilden und so Wasserkondensation aus der Umgebungsluft verringern. Durch das auf diese Weise veränderte Mikroklima wird das Wachstum von Schimmelpilzen auf den Bienen signifi kant reduziert und die Überlebenschancen der Bienenwolfnachkommen werden gesteigert. Diesen Nutzen der Beuteeinbalsamierung müssen sich die Weibchen allerdings mit einer verringerten zukünftigen Reproduktionsrate erkaufen. Larven von A. compressa entwickeln sich in Schaben wie der Amerikanischen Großschabe Periplaneta americana. Um sich gegen die zahlreichen schädlichen Keime auf den Schaben zu wehren, hygienisieren A. compressa-Larven ihre Wirte mit großen Mengen eines antimikrobiell wirksamen Sekrets. Diese spezielle Mischung von Substanzen besitzt Breitbandwirkung gegen die verschiedensten Mikroorganismen. Darüber hinaus setzen die Larven die Substanzen in einer räumlich wie zeitlich koordinierten Art und Weise, sowohl in Lösung als auch gasförmig, ein. Diese Strategie gewährleistet einen zuverlässigen Langzeitschutz durch drei Verteidigungslinien auch gegen Mikroben, die aus der Nestumgebung angreifen. Die hier gezeigten Beispiele verdeutlichen den Einfallsreichtum der Natur und die Mannigfaltigkeit möglicher Strate- gien im Kampf gegen Mikroben. Während sich bei Bienenwölfen die Mütter einen physikalischen Effekt zunutze machen, um Schimmelbildung auf der Larvalnahrung zu verhindern, sind es bei der Juwelwespe die Larven selbst, die mithilfe einer Mischung antimikrobieller Substanzen schädliche Mikroben auf ihren Wirten und in den Nestern bekämpfen. Zukünftige Untersuchungen der bisher kaum erforschten Fülle antimikrobieller Strategien von Insekten könnten neue Ansätze für den Kampf gegen Mikroorganismen in der Lebensmittelindustrie oder Humanmedizin liefern, um der Ausbreitung resistenter Mikroorganismen entgegenzuwirken.

Schlüsselwörter: Antimikrobielle Substanzen, antimykotische Substanzen, Antibiotika, Schimmel- pilze, Desinfektion

1. Introduction in exploring novel remedial measures and hygiene strategies with applications in hu- It is becoming increasingly evident that we man food industry or medicine. live in a microbial world (MCFALL-NGAI et al. Despite obvious differences between hu- 2013). While microorganisms play essential mans and insects, insects are equally defi ed roles in sustaining and promoting life, they by ubiquitous antagonistic microorganisms also act as pathogens or food competitors (e.g. TRIENENS et al. 2010; MCLEAN et al. and continuously challenge our survival and 2014). Since insects are the most successful well-being (JANZEN 1977; EUROPEAN FOOD group of eucaryotic organisms on earth SAFETY AUTHORITY 2011; WORLD HEALTH in terms of species numbers and diversity ORGANIZATION 2014). Countering the prob- as well as life history variety, they provide lems caused by detrimental microbes has be- nearly infi nite opportunities for the study come more and more diffi cult, owing to the of antimicrobial chemical compounds and emergence and rapid spread of resistance to strategies to ward off antagonistic microbes. the commonly used antimicrobials (WORLD Some groups can be considered es- HEALTH ORGANIZATION 2014). These dif- pecially prone to microbial attacks due to fi culties have stimulated the search for and their life-style or choice of habitat and food study of natural antimicrobials and antimi- resource. These insect species have evolved crobial strategies deployed in the natural elaborate and effective defense mechanisms environment (HAINE et al. 2008; ROZEN et to protect themselves, their offspring and al. 2008; DOSSEY 2010; POULSEN et al. 2011; their nutritional resources from microbial VILCINSKAS 2011; RÖHRICH et al. 2012). The pathogens and decomposers. They are thus results obtained from these studies may aid especially promising study objects for the Antimicrobial Defense Strategies in Two Solitary Wasp Species 3 discovery and exploration of antimicrobial tions from their contaminated food. Burying strategies deployed in natural systems. beetles of the Nicrophorus produce Social insects like , termites, bees, and and emit oral and anal exudates to preserve for example often live in highly the carcasses of small vertebrates that are dense colonies, thus facing an increased buried in soil and used as larval food sup- risk of pathogen transmission within the plies (ROZEN et al. 2008; JACQUES et al. 2009; group. Disease resistance mechanisms in COTTER et al. 2010; DEGENKOLB et al. 2011). social insects have been shown to include In this paper various aspects of antimicro- individual and social immunity (CREMER bial defense in two Hymenopteran model et al. 2007; CREMER & SIXT 2009; WILSON- systems, the Philanthus RICH et al. 2009; HAMILTON & BULMER triangulum (Hymenoptera: Crabronidae), a 2012; ROSENGAUS et al. 2013), as well as a ground-nesting digger wasp that has to cope so-called extended disease resistance (exo- with micro-climatic conditions favorable for symbiosis with Actinobacteria; CHOUVENC the rapid growth of mold fungi, and Ampu- et al. 2013). lex compressa (Hymenoptera: Ampulicidae), Furthermore, it has been proposed that the a cockroach that provides success of some invasive insect species can its offspring with high-risk food, will be be at least partly attributed to their ability reviewed. Since both species experience to mount strong anti-pathogen defenses. strong selective pressures by detrimental The invasive Asian ladybird or harlequin microbes they have evolved sophisticated ladybird beetle Harmonia axyridis, for ex- prophylactic mechanisms. ample, possesses a strong constitutive anti- microbial defense mechanism (the chemical 2. Prey embalming in the European compound Harmonine in the hemolymph; beewolf Philanthus triangulum GROSS et al. 2010; RÖHRICH et al. 2012) as well as an extended repertoire of inducible 2.1. Life history of Philanthus triangu- antimicrobial peptides (SCHMIDTBERG et al. lum 2013; VILCINSKAS et al. 2013). Insects living or nesting in soil, an envi- The European beewolf Philanthus trian- ronment tremendously rich in entomo- gulum provisions its offspring in under- pathogenic and opportunistic microbes, ground nests with paralyzed Western are under constant assault from diverse workers (Apis mellifera L., bacteria and fungi. Many of these soil- Hymenoptera, ) upon which their nesting insects provision their progeny larvae feed (e.g. STROHM 1995; STROHM & with highly nutritious food resources like LINSENMAIR 1997). Female P. triangulum pollen, prey or small verte- hunt and paralyze honeybees usually on brate carcasses that constitute attractive fl owers, bring them to a nest in fl ight (Fig. resources for competing, putrefactive 1), and deposit one to six prey bees in each microbes. Solitary bees from different brood cell of their nest as food for one genera line their subterranean brood cells . The larva hatches two to three days with hydrophobic substances from their after oviposition and feeds on the bees for Dufour’s glands to defend offspring and another fi ve to eight days before it spins provisions from fungi and bacteria (MI- a cocoon inside the brood cell in which CHENER 1964; CANE 1981 and references it usually overwinters. The size of the therein; HEFETZ et al. 1982). emerging adult beewolf depends on the Finally, insects that feed on microbe-laden amount of food, i.e. the number of bees, food resources are at risk for acquiring infec- provided by the mother (STROHM 2000).

Entomologie heute 26 (2014) 4 GUDRUN HERZNER

2.2. Benefi ts of prey embalming tion zone assays (HERZNER et al. 2007a). Surprisingly, even very high concentrations In a Philanthus triangulum nest, which may of the extracts failed to impede the growth consist of up to 34 brood cells that are of mold fungi, namely an Aspergillus strain subsequently provisioned via one main obtained from a beewolf brood cell, As- burrow (STROHM 1995), the majority of pergillus fumigatus, and Penicillium roquefortii, brood cells usually experience humid and in vitro, precluding a direct chemical effect warm conditions and the nutrient-rich of the surface extracts against mold fungi larval provisions are consequently highly (HERZNER et al. 2007a). susceptible to spoilage due to microbial But do female P. triangulum actually apply a degradation, mostly by mold fungi (Fig. secretion to their paralyzed prey bees during 2A). Fungal infestation of larval provisions “grooming”? A comparison of the chemical compromises larval survival (HERZNER et profi les on the cuticles of paralyzed, provi- al. 2011) and reduces the body size of the sioned honeybees from beewolf brood cells emerging adult beewolf (STROHM 2000). and paralyzed, but not provisioned control Beewolves should therefore have evolved bees by gas chromatography/mass spectro- defense mechanisms against fungal infes- metry revealed that provisioned bees carry tations of the provisions and their brood. on average signifi cantly higher amounts of In fact, only about 5% of the brood cells hydrocarbons than control bees (HERZNER et of the European beewolf are infested by al. 2007a; HERZNER & STROHM 2007, 2008). molds under natural conditions (STROHM Furthermore, while the chemical profi les & LINSENMAIR 2001), suggesting that of control bees are dominated by satu- beewolves employ effective counteractive rated hydrocarbons, the major compound measures in their fi ght against fungi. As P. on provisioned bees is the unsaturated triangulum is a mass-provisioning species hydrocarbon (Z)-(9)-pentacosene. As this and provides no active post-oviposition compound is also the major constituent of care to its progeny, any measure of prey a large head gland of P. triangulum females, preservation employed by the mother has the postpharyngeal gland (STROHM et al. to be taken prior to brood cell closure. 2007, 2008, 2010), it can be reasoned that The use of observation cages that allow females apply the secretion of this gland to the surveillance of beewolves inside their their honey bee prey during the “grooming” nests (STROHM & LINSENMAIR 1994/1995) behavior. revealed that female P. triangulum display a The postpharyngeal gland has long been conspicuous behavior: During excavating thought to be restricted to ants, where it a brood cell and before ovipositing on one serves several “social functions” such as of the bees in the brood cell, they repea- nestmate recognition (SOROKER et al. 1995; tedly and extensively lick the surface of all LUCAS et al. 2004; EELEN et al. 2006). P. bees (STROHM & LINSENMAIR 2001). As this triangulum is the fi rst solitary hymenopteran “grooming” behavior delays fungus growth species for which a PPG has been described on provisioned bees as compared to control (HERZNER et al. 2007b; STROHM et al. 2007). bees (Fig. 2B), STROHM & LINSENMAIR (2001) The morphology and ultrastructure of the proposed that female P. triangulum apply an beewolf postpharyngeal gland shows close antifungal secretion to the bees that retards resemblance to the postpharyngeal gland of mold growth. many species (STROHM et al. 2007, 2010). Following this hypothesis, surface extracts In P. triangulum females the postpharyngeal obtained from provisioned bees were tested gland contains mainly hydrocarbons, with for an antifungal activity by using inhibi- (Z)-9-pentacosene [or (Z)-9-heptacosene] Antimicrobial Defense Strategies in Two Solitary Wasp Species 5

Fig. 1: A female European beewolf, Philanthus triangulum, carrying its honeybee prey (Apis mellifera worker) in fl ight to its nest. Abb. 1: Ein Weibchen des Europäischen Bienenwolfs, Philanthus triangulum, bringt eine erbeutete Honigbiene (Apis mellifera-Arbeiterin) im Flug zum Nest.

Fig. 2: Honeybees after three days in artifi cial brood cells under conditions mirroring those in Philan- thus triangulum nests in the fi eld. A Control honeybee (paralyzed but not provisioned by P. triangulum) completely overgrown by mold fungi. B Paralyzed and provisioned bee almost devoid of mold fungi. Abb. 2: Honigbienen nach drei Tagen in künstlichen Brutzellen unter Bedingungen, wie sie in Philan- thus triangulum-Nestern im Freiland herrschen. A Kontrollbiene (von P. triangulum gelähmt, aber nicht verproviantiert), die völlig von Schimmelpilzen überwachsen ist. B Gelähmte und verproviantierte Biene, fast völlig frei von Schimmel.

Entomologie heute 26 (2014) 6 GUDRUN HERZNER being the major compound (STROHM et al. alternative explanation for the antifungal 2008). In addition to the hydrocarbons, the effect of prey “grooming” arises. gland contains some unusual long-chain A close examination of provisioned and saturated and unsaturated ketones. The fact not provisioned control bees revealed a that these ketones can also be found on con spicuous difference: While rather large provisioned bees (but not on conspecifi c water droplets were visible on control bees control bees; HERZNER et al. 2007a) is in (Fig. 3A), provisioned bees appeared line with the hypothesis that females apply mostly dry (Fig. 3B). Quantitative analyses their postpharyngeal gland secretion to the confi rmed a signifi cantly reduced water ac- surface of their prey. cumulation on provisioned bees (HERZNER In view of the above described fi ndings that & STROHM 2007). Further investigations (1) fungus germination as well as conidia of provisioned bees’ and control bees’ formation is signifi cantly retarded on pro- surfaces by scanning electron microscopy visioned bees carrying the postpharyngeal showed that female P. triangulum virtually gland secretion as compared to controls embalm their prey with hydrocarbons carrying simply their original epicuticular that form a thick and continuous coating hydrocarbons (STROHM & LINSENMAIR 2001; on the bee surface (Fig. 4) (HERZNER & HERZNER & STROHM 2007; HERZNER et al. STROHM 2007). This “food wrapping” 2011), but that (2) extracts of provisioned covers microstructures such as the sockets bees have no direct chemical effects on fungi of sensilla or tiny contaminants on the (HERZNER et al. 2007a), the question of an bee surface that could otherwise function

Fig. 3: Stereomicroscopic image of forewing of A a control honeybee (paralyzed but not provisioned by Philanthus triangulum), and B a paralyzed and provisioned honeybee, after three days in an artifi cial brood cell under conditions mirroring those in beewolf brood cells in the fi eld. Note the rather large water droplets on the wing of the control bee that have formed due to conden- sation. By contrast, the surface of the provisioned bee appears mostly dry. Abb. 3: Stereomikroskopische Auf- nahme des Vorderflügels A einer Kontroll-Honigbiene (gelähmt, aber nicht verproviantiert von Philanthus triangulum), und B einer gelähmten und verproviantierten Honigbiene, nach drei Tagen in künstlichen Brut- zellen unter Bedingungen, wie sie in Bienenwolfnestern im Freiland herr- schen. Die großen Wassertropfen auf der Kontrollbiene haben sich durch Kondensation geformt. Die Oberfl ä- che der verproviantierten Biene ist im Gegensatz dazu weitgehend trocken. Antimicrobial Defense Strategies in Two Solitary Wasp Species 7 as nucleation sites for water condensation COTTER et al. 2010; DEGENKOLB et al. 2011). or could promote infectious processes by However, the indirect, physically mediated, fungi (BOUCIAS & PENDLAND 1991; BEYSENS mechanism of prey preservation by P. tri- 1995; SOSA-GOMEZ et al. 1997). Moreover, angulum represents a unique adaptation of the embalming increases the proportion of higher in their combat with fungi. unsaturated hydrocarbons on the bee surface about threefold so that the cuticular coating 2.3. Costs of prey embalming forms an oily, hydrophobic layer (HERZNER & STROHM 2007). The resulting topographic Female Philanthus triangulum allocate a and physico-chemical changes of the bee substantial share of their resources to hy- surface heavily reduce water condensation drocarbon production for prey embalming on the bees. The antifungal effect of prey (HERZNER & STROHM 2008; HERZNER et al. embalming is thus, surprisingly, mediated 2011). It is unequivocal that the parental care by a physical effect: The reduced availability behavior of prey embalming is benefi cial of water renders microclimatic conditions in that it reduces fungal infestations of the on embalmed bees unsuitable for fungal larval provisions and enhances offspring germination and growth. Other insects, like survival (STROHM & LINSENMAIR 2001; HERZ- e.g. burying beetles of the genus Nicrophorus, NER & STROHM 2007; HERZNER et al. 2011). are also known to preserve larval provisions However, when studying parental care be- (ROZEN et al. 2008; JACQUES et al. 2009; haviors, both benefi ts as well as costs to the

Fig 4: Scanning electron micrograph of a honeybee embalmed by a Philanthus triangulum female (scale bar = 10 μm). The surface is coated with a continuous, oily layer of unsaturated hydrocarbons. Abb. 4: Rasterelektronenmikroskopische Aufnahme einer Honigbienenantenne, die von einem Philanthus triangulum Weibchen einbalsamiert wurde (Maßstabsleiste = 10 μm). Die Oberfl äche ist durchgehend mit einer öligen Schicht aus ungesättigten Kohlenwasserstoffen überzogen.

Entomologie heute 26 (2014) 8 GUDRUN HERZNER providing individual have to be considered. to balance their investment of hydrocarbons A quantifi cation of hydrocarbons on freshly in current and future offspring. provisioned prey bees obtained from obser- vation cages revealed that the mean amount 2.4. Summary and Outlook: Prey em- of postpharyngeal gland secretion a female balming in Philanthus triangulum allocates to a given brood cell increases with the number of bees in the brood cell (HERZ- The above described example shows that NER et al. 2011). Brood cells containing many mold fungi may be warded off by other bees are thus costlier to produce in terms of means than antifungals that chemically in- prey embalming than brood cells containing terfere with their germination and growth. fewer bees. Interestingly, the amount of se- By embalming their prey bees with large cretion per single bee decreases signifi cantly amounts of unsaturated hydrocarbons, with an increasing number of bees in the female Philanthus triangulum alter the micro- brood cell, suggesting that the actual avail- climatic conditions on the bee surface and ability of the postpharyngeal gland secretion by doing so deprive fungi of the water they is limited (HERZNER et al. 2011). need to thrive. The ultimate evolutionary When females are experimentally forced to and ecological signifi cance of the parental upregulate their embalming effort for one care behavior of prey embalming with the brood cell, the amount of postpharyngeal postpharyngeal gland secretion is beyond gland secretion applied to the subsequent doubt. If beewolf females allocate their brood cell is reduced (HERZNER et al. 2011). resources accordingly, they can increase the This reduction in the amount of postpha- survival or fi tness of their progeny and, ryngeal gland secretion applied to the bees thus, their own reproductive output under results in an elevated risk of fungal infesta- conditions that could otherwise cause high tions and, thus, in higher larval mortality and mortality or reduced progeny size (STROHM reduced reproductive output of the beewolf 2000; HERZNER et al. 2011). Recently, it has mother (HERZNER et al. 2011). been shown that females of the neotropical P. triangulum females that allocate large beewolf genus Trachypus also possess post- amounts of secretion for one brood cell pharyngeal glands and embalm their prey have two options: They can postpone the bees (HERZNER et al. 2013a). next brood cell until the hydrocarbon stores Remarkably, the European beewolf P. trian- in the postpharyngeal gland are replenished gulum has evolved one other intriguing and and incur a reduced rate of reproduction. effective antimicrobial defense mechanism: Or they can allocate smaller amounts of Its cocoons are protected by a cocktail of hydrocarbons to the subsequent brood cell antibiotics produced by symbiotic Streptomy- with the consequence that these bees will ces bacteria (KALTENPOTH et al. 2005, 2014; have a higher risk of becoming moldy. In KROISS et al. 2010; KOEHLER et al. 2013). This each case, the embalming of the bees for protection is highly benefi cial in terms of the current offspring reduces the future microbe suppression within the brood cells reproductive success. Prey embalming in P. (KALTENPOTH et al. 2005; KROISS et al. 2010). triangulum hence qualifi es as parental invest- Since the bacterial symbionts are maintained ment in the sense of TRIVERS (1972) in that in specialized antennal glands of females it provides benefi ts for current offspring but (GOETTLER et al. 2007), this antimicrobial incurs costs in terms of a decrease in the defense mechanism might as well be costly. parent’s future reproductive ability (HERZ- It will be interesting to see whether these two NER et al. 2011). To maximize their lifetime mechanisms interact in their action against reproductive success female beewolves have microbes and whether the expenditures for Antimicrobial Defense Strategies in Two Solitary Wasp Species 9 the two components of parental care are surrounded by the cocoon and the cock- somehow correlated. In any case, owing to roach cuticle (WEISS et al. 2014). the two antimicrobial defense mechanisms, beewolf offspring are protected from the 3.2. Host sanitation by Ampulex com- egg stage on until emergence of the adult pressa larvae wasp (STROHM & LINSENMAIR 2001; HERZNER & STROHM 2007; KOEHLER et al. 2013). Cockroaches like Periplaneta americana are known to pick-up, carry and transfer 3. Antimicrobial defense in larvae of microorganisms from their environment the Emerald cockroach wasp Ampulex (Figure 6). Microorganisms isolated from compressa cockroaches include various entomo- und human-pathogenic strains like e.g. Serra- 3.1. Life history of Ampulex compressa tia marcescens, Escherichia coli, Staphylococcus aureus, Aspergillus fl avus, Aspergillus nomius, The emerald cockroach wasp Ampulex and Candida sp. (CHAICHANAWONGSAROJ et compressa is, like Philanthus triangulum, a al. 2004; PAI et al. 2004, 2005; LEMOS et al. parasi toid wasp sensu lato that stores insect 2006; FAKOORZIBA et al. 2010; HERZNER et prey for future consumption by its larvae al. 2013b; WEISS et al. 2014). Since Ampulex (WILLIAMS 1942; HEITMANS 1990; LIBERSAT compressa offspring spent the whole deve- 2003; KEASAR et al. 2006; LIBERSAT & GAL lopmental phase on their cockroach hosts, 2014; WEISS et al. 2014). It depends on which concomitantly are their sole food cockroaches like the peridomestic Ameri- source, these wasps have to deploy effective can cockroach Periplaneta americana (Blat- hygienic measures against microbes. taria, Blattidae) as larval food. Female A. As there were no indications of any prey compressa catch cockroaches (Fig. 5) and put preservation behavior by A. compressa mo- them into a lethargic state by injecting their thers, it seemed reasonable to assume that into the supraesopharyngeal gang- the larvae themselves exert some kind of lion (“”) of the cockroach (LIBERSAT antimicrobial defense. By carefully cutting & GAL 2014). The docile cockroach is then holes in the abdomens of parasitized guided to a nest, where the female attaches cockroaches and covering these holes with one single egg to the coxa of one of the glass cover slips A. compressa larvae can be mesothoracic legs. The female subsequently monitored through a “window” inside their closes the nest with twigs, leaves, stones etc. cockroach hosts (HERZNER et al. 2013b). and thus virtually immures the cockroach Such observations disclosed that A. compressa inside the nest. When the egg hatches, the larvae secrete a clear liquid with which they larva remains at the oviposition site on the impregnate their cockroach hosts (Fig. 7A; outer cockroach cuticle for six to seven HERZNER et al. 2013b). Chemical analyses days and drinks hemolymph of the still revealed that this secretion contains nine living host through a hole in the cuticle. relatively polar compounds, mainly (R)-(- Subsequently, the larva migrates inside )-mellein [(R)-(-)-3,4-dihydro-8-hydroxy- the cockroach to feed on the inner tissues, 3-methylisocoumarin] and micromolide causing the death of the host. After eroding [(4R,9Z)-octadec-9-en-4-olide] (Fig. 7B), and the cockroach completely the larva spins some hydrocarbons (HERZNER et al. 2013b). its cocoon inside the then empty cock- The amounts of antimicrobials applied by a roach carcass. Until its emergence as adult single larva to its cockroach host are aston- wasp about six weeks after oviposition (at ishingly large (about 5 mg; HERZNER et al. 27 °C) the developing individual remains 2013b; WEISS et al. 2014).

Entomologie heute 26 (2014) 10 GUDRUN HERZNER

Fig. 5: A female emerald cockroach wasp, Ampulex compressa, with its cockroach prey, Periplaneta americana. Abb. 5: Ein Weibchen der Juwelwespe Ampulex compressa mit ihrer Schabenbeute, einer Periplaneta americana.

Fig. 6: A Periplaneta americana on nutrient agar in a Petri dish. Note the red bacterial colonies (Serratia marc- escens) and the mold fungi (Aspergillus sp.) emerging from the cockroach and grow- ing on the agar. Abb. 6: Eine Periplaneta americana auf Nähragar in einer Petrischale. Rote Bakterienkoloni- en (Serratia marcescens) und Schimmelpilze (Aspergilllus sp.) wach- sen von der Schabe ausgehend auf dem Agar. Antimicrobial Defense Strategies in Two Solitary Wasp Species 11

Antimicrobial assays revealed that the polar can be attributed mainly to the activity of fraction of the secretion is active against (R)-(-)-mellein. The effect against S. hyicus the Gram-negative, entomopathogenic could only be explained by the combined bacterium S. marcescens, which had previ- activity of (R)-(-)-mellein and micromolide. ously been isolated from P. americana cock- In addition to the two major compounds roaches, as well as against the Gram-positive (R)-(-)-mellein and micromolide the secreti- Staphylococcus hyicus (HERZNER et al. 2013b). on contains smaller amounts of the mellein Similar assays with synthetic (R)-(-)-mellein derivatives 7-hydroxymellein, 4-hydroxymel- and micromolide showed that the activity lein, and 5-hydroxymellein, the γ-lactones of the larval secretion against S. marcescens (R)-hexadecan-4-olide, heptadecan-4-olide,

Fig. 7: A A larva of Ampulex compressa inside its Periplaneta americana host applying droplets of anti- microbial secretion (arrows) to a cover slip that covers an artifi cial opening in the abdominal cuticle of the cockroach. B Chemical structures of the two major constituents of the antimicrobial larval secretion, the methylisocoumarin (R)-(-)-mellein and γ-lactone micromolide. Abb. 7: A Ampulex compressa-Larve im Inneren ihrer Wirtsschabe Periplaneta americana bei der Abgabe des antimikrobiellen Sekrets (Pfeile) auf ein Deckgläschen, das ein künstliches Loch im Abdomen der Wirtsschabe abdeckt. B Strukturformeln der beiden Hauptbestandteile des antimikrobiellen Larvalsekrets, des Methylisocumarins (R)-(-)-Mellein und des γ-Laktons Micromolid.

Entomologie heute 26 (2014) 12 GUDRUN HERZNER octadeca-9,12-dien-4-olide, and (R)-octade- of the cockroach by virtually soaking it can-4-olide (HERZNER et al. 2013b; WEISS et with their antimicrobial secretion to create al. 2014). As (R)-(-)-mellein, micromolide a germfree environment for cocoon spin- and the other constituents of the secretion ning (WEISS et al. 2014). After successful of A. compressa larvae seem to have anti- cocoon formation A. compressa progeny microbial activity against a wide range of must protect themselves against opportu- different microbes like Gram-negative and nistic microbes that may invade from the Gram-positive bacteria, mycobacteria, fungi, nest surroundings during the pupal phase and a virus (SCHULZ et al. 1995; KROHN et al. and metamorphosis. The antimicrobials 1997; HÖLLER et al. 1999; DAI et al. 2001; EL- are therefore also incorporated into the co- MEHALAWY et al. 2005; MA et al. 2005; YUAN coon walls to establish a second defensive et al. 2008; RUKACHAISIRIKUL et al. 2009; barrier against microbes in addition to the FENG et al. 2010; OLIVEIRA et al. 2011; ZHAO cockroach shell (WEISS et al. 2014). Inte- et al. 2012; HERZNER et al. 2013b; WEISS et restingly, the antimicrobial profi les of the al. 2014), it can be reasoned that A. compressa two protective layers surrounding the wasp larvae sanitize their cockroach hosts with a offspring, the cockroach and the cocoon, blend of antimicrobials that provide reliable differ markedly with cockroaches bearing broad-spectrum antimicrobial activity. higher proportions of (R)-(-)-mellein and The microbial hazards developing A. cocoons bearing higher proportions of compressa immatures may encounter are micromolide. manifold. Investigating the antimicrobial Studies on the persistence of the anti- strategy of the wasp larvae in more detail microbials on the parasitized cockroaches revealed that they have evolved a sophisti- and cocoons revealed that the amount of cated and intriguing strategy to face these the non-volatile micromolide remains rather diverse threats (HERZNER et al. 2013b; constant after the cocoon has been comple- WEISS et al. 2014). The fi rst danger certainly ted, whereas about 80% of the volatile (R)- emerges from the cockroach host itself, (-)-mellein evaporate from the cockroach as cockroaches represent a high risk food surface and accumulate in the nest space naturally contaminated with pathogenic (WEISS et al. 2014). Microbial challenge as- and putrefactive microbes (CHAICHANA- says revealed that the gaseous (R)-(-)-mellein WONGSAROJ et al. 2004; PAI, et al. 2004, 2005; surrounding the parasitized cockroach LEMOS et al. 2006; FAKOORZIBA et al. 2010; hampers the growth of entomopathogenic HERZNER et al. 2013b; WEISS et al. 2014). and opportunistic bacteria (S. marcescens) and Chemical analyses of different develop- fungi (Aspergillus sydowii, Metarhizium brunne- mental stages of A. compressa and their host um) in A. compressa nests (WEISS et al. 2014). cockroaches showed that larvae start sani- Owing to its volatility (R)-(-)-mellein can tizing their food with their antimicrobial permeate the whole nest space and affect mi- secretion as soon as they have entered their crobes at some distance from the cockroach host to feed on its inner tissues (HERZNER and wasp offspring as well as those hidden et al. 2013b; WEISS et al. 2014). In this way in otherwise inaccessible places. Sanitizing the larvae prevent decomposition of their the nest space with vaporous (R)-(-)-mellein sole food source as well as infection by thus represents a kind of front line defense. food borne microbes. Subsequently, the (R)-(-)-mellein seems to have a triple func- larvae have to take care that no detrimental tion as food preservative during feeding, microbes end up inside the cocoon, where protective antimicrobial cover on cockroach they could harm or kill them. The larvae shell and cocoon, as well as fumigant of the therefore thoroughly disinfect the inside nest space. Antimicrobial Defense Strategies in Two Solitary Wasp Species 13

3.3 Summary and Outlook: Host sani- gens and food competitors, because these tation by Ampulex compressa larvae microbes compromise their own and their offspring’s survival and well-being. As an The synthesis and secretion of antimicro- adaptive response to these pressures insects bials is without doubt crucial to the survi- have evolved potent antimicrobial measures. val and well-being of many insect species. Since the hazards that the diverse insect The specifi c combination of substances species experience during their ontogenies in an antimicrobial blend and the way in are manifold, so are the remedies that have which they are deployed are likely to be evolved to rise up to these challenges. critical to the success of an antimicrobial In both beewolves and cockroach wasps, the strategy. specifi c life-style of the larvae entails certain In the emerald cockroach wasp Ampulex risks in terms of adverse effects by microbes. compressa, the larvae apply a cocktail of Beewolves are ground-nesting species that nine different substances to their hosts store Hymenopteran prey under micro climatic and cocoons, most of which have proven conditions that favor fungal growth. To over- antimicrobial activity (SCHULZ et al. 1995; come these microbial threats the beewolf KROHN et al. 1997; HÖLLER et al. 1999; DAI mothers preserve the larval food by applying et al. 2001; EL-MEHALAWY et al. 2005; MA et copious amounts of oily lipids. They aim at al. 2005; YUAN et al. 2008; RUKACHAISIRIKUL the Achilles heel of most fungi and make use et al. 2009; FENG et al. 2010; OLIVEIRA et of the physical effect of reduced water con- al. 2011; ZHAO et al. 2012; HERZNER et al. densation to retard fungal growth. 2013b; WEISS et al. 2014). As a result of The cockroach wasp choses food resources the spatially and temporally coordi nated for its offspring which have a high probabil- deployment of several antimicrobials ity of being contaminated with detrimental with combined antimicrobial activity and microbes. The larvae themselves practice different physico-chemical properties, de- food hygiene in that they impregnate veloping A. compressa are protected by three their hosts with large amounts of a blend lines of defense until adult emergence: the of several antimicrobial compounds that cocoon and cockroach cuticle impregna- directly interfere with bacterial and fungal ted with antimicrobials and the vaporous growth. Moreover, the larvae implement (R)-(-)-mellein in the surrounding nest the different antimicrobials in a spatially and space. This multifaceted defense strategy temporally coordinated manner and mount has apparently evolved to match the need three lines of defense to secure their survival for a dependable and enduring protection during the especially vulnerable stages of against an unpredictable spectrum of detri- their development. mental microbes. The excretion and usage The examples described here clearly dem- of antimicrobials outside their bodies is onstrate the sophistication and ingenuity without much doubt only one facet of the of nature’s solutions to microbial hazards. immune defense strategy of A. compressa Considering that Philanthus triangulum and larvae. Future studies will most likely reveal Ampulex compressa are only two out of a other components of immune defense in myriad of species of insects on earth, most these wasps. of which have not been investigated as yet, it becomes clear what a cornucopia of un- 4. Overall Conclusion discovered antimicrobial strategies the insect world still is. And even though research on Like humans, insects face strong selective these topics is fascinating and worthwhile by pressures to cope with microbial patho- itself, it might well be that the antimicrobial

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