Evolutionary stability of antibiotic protection in a defensive symbiosis.

The increasing resistance of human pathogens severely limits the efficacy of antibiotics in medicine, yet many animals, including solitary beewolf wasps, successfully engage in defensive alliances with antibiotic-producing bacteria for millions of years. Here, we report on the in situ production of 49 derivatives belonging to three antibiotic compound classes (45 piericidin derivatives, 3 streptochlorin derivatives, and nigericin) by the symbionts of 25 beewolf host species and subspecies, spanning 68 million years of evolution. Despite a high degree of qualitative stability in the antibiotic mixture, we found consistent quantitative differences between species and across geographic localities, presumably reflecting adaptations to combat local pathogen communities. Antimicrobial bioassays with the three main components and in silico predictions based on the structure and specificity in polyketide synthase domains of the piericidin biosynthesis gene cluster yield insights into the mechanistic basis and ecoevolutionary implications of producing a complex mixture of antimicrobial compounds in a natural setting.

Striking cuticular hydrocarbon dimorphism in the mason wasp Odynerus spinipes and its possible evolutionary cause (Hymenoptera: Chrysididae, Vespidae).

Cleptoparasitic wasps and bees smuggle their eggs into the nest of a host organism. Here the larvae of the cleptoparasite feed upon the food provision intended for the offspring of the host. As cleptoparasitism incurs a loss of fitness for the host organism (offspring of the host fail to develop), hosts of cleptoparasites are expected to exploit cues that alert them to potential cleptoparasite infestation. Cuticular hydrocarbons (CHCs) could serve as such cues, as insects inevitably leave traces of them behind when entering a nest. By mimicking the host's CHC profile, cleptoparasites can conceal their presence and evade detection by their host. Previous studies have provided evidence of cleptoparasites mimicking their host's CHC profile. However, the impact of this strategy on the evolution of the host's CHC profile has remained unexplored. Here, we present results from our investigation of a host-cleptoparasite system consisting of a single mason wasp species that serves syntopically as the host to three cuckoo wasp species. We found that the spiny mason wasp (Odynerus spinipes) is able to express two substantially different CHC profiles, each of which is seemingly mimicked by a cleptoparasitic cuckoo wasp (i.e. Chrysis mediata and Pseudospinolia neglecta). The CHC profile of the third cuckoo wasp (Chrysis viridula), a species not expected to benefit from mimicking its host's CHC profile because of its particular oviposition strategy, differs from the two CHC profiles of its host. Our results corroborate the idea that the similarity of the CHC profiles between cleptoparasitic cuckoo wasps and their hosts are the result of chemical mimicry. They further suggest that cleptoparasites may represent a hitherto unappreciated force that drives the evolution of their hosts' CHCs.

Morphology, chemistry and function of the postpharyngeal gland in the South American digger wasps Trachypus boharti and Trachypus elongatus.

Microbes pose severe threats to animals as competitors or pathogens and strongly affect the evolution of life history traits like parental care. Females of the European beewolf Philanthus triangulum, a solitary digger wasp, provision their offspring with paralyzed honeybees and embalm them with the secretion from large postpharyngeal glands (PPG) that contain mainly unsaturated hydrocarbons. This coating changes the physico-chemical properties of the prey surface, causes a reduction of water condensation and retards growth of mold fungi. Here we examined the closely related South American genus Trachypus, which shows a life-history similar to Philanthus. We investigated whether Trachypus spp. also possess PPGs and embalm larval provisions. Using histological methods and 3D reconstructions we show that Trachypus boharti and T. elongatus possess PPGs that are similar to P. triangulum but somewhat smaller. The ultrastructure of the gland epithelium suggests that the gland content is at least partly sequestered from the hemolymph. Chemical analyses using gas chromatography / mass spectrometry revealed that both the cuticle and PPGs of Trachypus contain mainly unsaturated long-chain hydrocarbons. The gland of T. boharti additionally contains long-chain ketones. The hydrocarbons from the PPG of T. elongatus occurred on prey bees excavated from nests in the field but not on conspecific control bees. While the embalming only slightly elevated the amount of hydrocarbons on prey bees, the proportion of unsaturated hydrocarbons, which is crucial for the antifungal effect, was significantly increased. The Trachypus species under study possess PPGs that are very similar to the PPG of P. triangulum with regard to morphology, ultrastructure and chemistry. Moreover, we provide clear evidence that T. elongatus females embalm their prey, presumably as a means of prey preservation. The observed differences among Trachypus and Philanthus in gland size and prey embalming may have evolved in response to divergent ecological conditions.

Cuticular hydrocarbons of the South American fruit fly Anastrepha fraterculus: variability with sex and age.

Insect cuticular hydrocarbons are usually species-specific mixtures and may serve for species and gender recognition. They are, therefore, widely used in the chemotaxonomy and zoogeography of various insect taxa. In order to provide a basic study for further comparative analyses of cuticular hydrocarbon (CHC) profiles of cryptic species hidden within the South American fruit fly Anastrepha fraterculus complex (Diptera: Tephritidae), we analyzed the composition of the CHCs and their production with respect to age and sex in a laboratory population from Tucuman, Argentina. Several techniques of gas chromatography with mass spectrometric detection have been used in order to develop a suitable method for CHC identification, i.e., GC-MS in EI mode, GC-MS in CI mode, and GC×GC/TOFMS. Our analyses revealed a complex profile of aliphatic hydrocarbons in both males and females, consisting predominantly of n-alkanes, methyl-branched alkanes, as well as of alkenes and alkadienes. In young individuals (up to about 5 days after emergence), the CHC profiles were similar in males and females. However, in older flies, these profiles diverged and became clearly sex-specific. The temporal dynamics of the CHC patterns in both sexes were evaluated using multivariate exploratory techniques.

Host plant use by competing acacia-ants: mutualists monopolize while parasites share hosts.

Protective ant-plant mutualisms that are exploited by non-defending parasitic ants represent prominent model systems for ecology and evolutionary biology. The mutualist Pseudomyrmex ferrugineus is an obligate plant-ant and fully depends on acacias for nesting space and food. The parasite Pseudomyrmex gracilis facultatively nests on acacias and uses host-derived food rewards but also external food sources. Integrative analyses of genetic microsatellite data, cuticular hydrocarbons and behavioral assays showed that an individual acacia might be inhabited by the workers of several P. gracilis queens, whereas one P. ferrugineus colony monopolizes one or more host trees. Despite these differences in social organization, neither of the species exhibited aggressive behavior among conspecific workers sharing a tree regardless of their relatedness. This lack of aggression corresponds to the high similarity of cuticular hydrocarbon profiles among ants living on the same tree. Host sharing by unrelated colonies, or the presence of several queens in a single colony are discussed as strategies by which parasite colonies could achieve the observed social organization. We argue that in ecological terms, the non-aggressive behavior of non-sibling P. gracilis workers--regardless of the route to achieve this social structure--enables this species to efficiently occupy and exploit a host plant. By contrast, single large and long-lived colonies of the mutualist P. ferrugineus monopolize individual host plants and defend them aggressively against invaders from other trees. Our findings highlight the necessity for using several methods in combination to fully understand how differing life history strategies affect social organization in ants.

Queen pheromones in Temnothorax ants: control or honest signal?

BACKGROUND: The division of reproductive labor among group members in insect societies is regulated by "queen pheromones". However, it remains controversial whether these are manipulative, i.e., actively suppress worker reproduction, or honestly signal the fertility status of the queen to which workers react in their own interest by refraining from laying eggs. Manipulative queen control is thought to lead to an evolutionary arms race between queens and workers, resulting in complex queen bouquets that diverge strongly among different populations and species. In contrast, honest signals would evolve more slowly and might therefore differ less strongly within and among species. RESULTS: We aimed at determining the tempo of the evolution of queen signals in two ways. First, we investigated whether queens of Temnothorax ants are capable of controlling egg laying by workers of their own, closely, and distantly related species. Second, we compared the species- and caste-specific patterns of cuticular hydrocarbons, which are assumed to convey information on reproductive status. In mixed-species colonies, queens were not able to fully suppress egg-laying and male production by workers of unrelated species, while workers did not reproduce under the influence of a queen from their own species. Furthermore, the chemical profiles differed more strongly among queens of different species than among the respective workers. CONCLUSIONS: Our results suggest that cuticular hydrocarbons associated with fecundity are not fully conserved in evolution and evolve slightly faster than worker-specific components in the blend of cuticular hydrocarbons. While this higher rate of evolution might reflect an arms race between queens and workers, the observation that workers still respond to the presence of a queen from another species support the honest signal hypothesis. Future studies need to examine alternative explanations for a higher rate of evolution of queen-specific substances, such as an involvement of such compounds in mating.

Rapid identification of insect cuticular hydrocarbons using gas chromatography-ion-trap mass spectrometry.

Insect cuticular hydrocarbons (CHCs) are not only essential for desiccation resistance, they also play an important role as chemical signals and cues in social as well as solitary insects. The identification of CHCs is, therefore, crucial to an understanding of the chemical communication within and between insect species. We describe a method for rapid, simple, and unambiguous identification of CHCs using gas chromatography-ion-trap mass spectrometry. External ionization configuration in combination with a low ion-trap temperature resulted in dramatically increased intensities of molecular ions for alkanes, alkenes and alkadienes, and in high-mass fragmentation patterns with intense ions characteristic for methyl-branched hydrocarbons comparable to those obtained with quadrupole instruments. Additionally, we present an external chemical ionization-tandem mass-spectrometric method that allows for the determination of double-bond positions in alkenes and alkadienes without the need for derivatization prior to analysis.

Symbiotic Streptomycetes provide antibiotic combination prophylaxis for wasp offspring.

Beewolf digger wasps cultivate specific symbiotic bacteria (Streptomyces spp.) that are incorporated into the larval cocoon for protection against pathogens. We identified the molecular basis of this protective symbiosis in the natural context and demonstrate that the bacteria produce a 'cocktail' of nine antibiotic substances. The complementary action of all symbiont-produced antibiotics confers a potent antimicrobial defense for the wasp larvae that parallels the 'combination prophylaxis' known from human medicine.

An epicuticular multilayer reflector generates the iridescent coloration in chrysidid wasps (Hymenoptera, Chrysididae).

Chrysidid wasps in the subfamily Chrysidinae are brood parasitoids or cleptoparasites of other insects and famous for their cuticular iridescence. In this study, we examine the dorsal abdominal cuticle of the chrysidid wasp Hedychrum rutilans to identify the underlying color mechanism. Using scanning electron microscopy, reflectance spectral analysis, and theoretical calculations, we demonstrate the presence of an epicuticular multilayer reflector consisting of six lamellae with a thickness of 185 nm each. The lamellae exhibit a rough surface probably functioning as spacers between the individual layers. The reflector has a measured reflectance maximum at lambda = 630 nm, i.e., in the red part of the visible spectrum of light at normal incidence and the reflectance maximum shifts to green as the angle of incidence increases. Complementary theoretical modeling corroborates the view that the epicuticular multilayer generates the iridescent color of the chrysidid cuticle.

Phylogeny and phenotypic characterization of pathogenic Cryptococcus species and closely related saprobic taxa in the Tremellales.

The basidiomycetous yeasts Cryptococcus neoformans and Cryptococcus gattii are closely related sibling species that cause respiratory and neurological disease in humans and animals. Within these two recognized species, phylogenetic analysis reveals at least six cryptic species defined as molecular types (VNI/II/B, VNIV, VGI, VGII, VGIII, and VGIV) that comprise the pathogenic Cryptococcus species complex. These pathogenic species are clustered in the Filobasidiella clade within the order Tremellales. Previous studies have shown that the Filobasidiella clade also includes several saprobic fungi isolated from insect frass, but information evaluating the relatedness of the saprobes and pathogens within this cluster is limited. Here, the phylogeny encompassing a subset of species in the Tremellales lineage that clusters closely with the pathogenic Cryptococcus species complex was resolved by employing a multilocus sequencing approach for phylogenetic analysis. Six highly conserved genomic loci from 15 related basidiomycete species were sequenced, and the alignments from the concatenated gene sequences were evaluated with different tree-building criteria. Furthermore, these 15 species were subjected to virulence and phenotype assays to evaluate their pathogenic potential. These studies revealed that Cryptococcus amylolentus and Tsuchiyaea wingfieldii, two nonpathogenic sibling species, are the taxa most closely related to the pathogens C. neoformans and C. gattii and together with Filobasidiella depauperata form a Cryptococcus sensu stricto group. Five other saprobic yeast species form the Kwoniella clade, which appears to be a part of a more distantly related sensu lato group. This study establishes a foundation for future comparative genomic approaches that will provide insight into the structure, function, and evolution of the mating type locus, the transitions in modes of sexual reproduction, and the emergence of human pathogenic species from related or ancestral saprobic species.

Scent of a queen-cuticular hydrocarbons specific for female reproductives in lower termites.

In social insects, it is assumed that signals of the queen inform nestmates about her reproductive status. Thus, workers forego their own reproduction if the queen signals high fertility. In hemimetabolous termites, little is known about reproductive inhibition, but evidence exists for a royal-pair control. Workers of lower termites exhibit a high developmental flexibility and are potentially able to become reproductives, but the presence of a fertile reproductive restrains them from reaching sexual maturity. The nature of this control, however, remains unknown. Here, we report on qualitative differences in cuticular hydrocarbon profiles between queens and workers of the basal drywood termite Cryptotermes secundus. Queens were characterized by a shift to long-chained and branched hydrocarbons. Most remarkably, similar chemical patterns are regarded as fertility cues of reproductives in social Hymenoptera. This might suggest that both groups of social insects convergently evolved similar chemical signatures. The present study provides deeper insights into how termites might have socially exploited these signatures from sexual communication in their cockroach-like ancestor.

Chemical correlates of reproduction and worker policing in a myrmicine ant.

In a number of wasps, bees, and in particular ponerine ants, quantitative and qualitative variation in the profile of cuticular hydrocarbons is associated with variation in fecundity and is likely to serve for communicating the reproductive status of an individual. Here, we demonstrate that the chemical profile on the cuticle of fertile workers and queens of the myrmicine ant Temnothorax unifasciatus is different from that of non-reproductive workers. Fertility and apparently also cuticular signatures are reversible under the influence of policing by worker aggression. Though no policing by egg-eating occurs in this species, queen and worker laid eggs also differed in their chemical profile.

A cuckoo in wolves' clothing? Chemical mimicry in a specialized cuckoo wasp of the European beewolf (Hymenoptera, Chrysididae and Crabronidae).

BACKGROUND: Host-parasite interactions are among the most important biotic relationships. Host species should evolve mechanisms to detect their enemies and employ appropriate counterstrategies. Parasites, in turn, should evolve mechanisms to evade detection and thus maximize their success. Females of the European beewolf (Philanthus triangulum, Hymenoptera, Crabronidae) hunt exclusively honeybee workers as food for their progeny. The brood cells containing the paralyzed bees are severely threatened by a highly specialized cuckoo wasp (Hedychrum rutilans, Hymenoptera, Chrysididae). Female cuckoo wasps enter beewolf nests to oviposit on paralyzed bees that are temporarily couched in the nest burrow. The cuckoo wasp larva kills the beewolf larva and feeds on it and the bees. Here, we investigated whether H. rutilans evades detection by its host. Since chemical senses are most important in the dark nest, we hypothesized that the cuckoo wasp might employ chemical camouflage. RESULTS: Field observations suggest that cuckoo wasps are attacked by beewolves in front of their nest, most probably after being recognized visually. In contrast, beewolves seem not to detect signs of the presence of these parasitoids neither when these had visited the nest nor when directly encountered in the dark nest burrow.In a recognition bioassay in observation cages, beewolf females responded significantly less frequently to filter paper discs treated with a cuticular extract from H. rutilans females, than to filter paper discs treated with an extract from another cuckoo wasp species (Chrysis viridula). The behavior to paper discs treated with a cuticular extract from H. rutilans females did not differ significantly from the behavior towards filter paper discs treated with the solvent only.We hypothesized that cuckoo wasps either mimic the chemistry of their beewolf host or their host's prey. We tested this hypothesis using GC-MS analyses of the cuticles of male and female beewolves, cuckoo wasps, and honeybee workers. Cuticle extracts of Hedychrum nobile (Hymenoptera: Chrysididae) and Cerceris arenaria (Hymenoptera: Crabronidae) were used as outgroups. There was little congruence with regard to cuticular compounds between H. rutilans females and honeybees as well as females of C. arenaria and H. nobile. However, there was a considerable similarity between beewolf females and H. rutilans females. Beewolf females show a striking dimorphism regarding their cuticular hydrocarbons with one morph having (Z)-9-C25:1 and the other morph having (Z)-9-C27:1 as the major component. H. rutilans females were more similar to the morph having (Z)-9-C27:1 as the main component. CONCLUSION: We conclude that H. rutilans females closely mimic the composition of cuticular compounds of their host species P. triangulum. The occurrence of isomeric forms of certain compounds on the cuticles of the cuckoo wasps but their absence on beewolf females suggests that cuckoo wasps synthesize the cuticular compounds rather than sequester them from their host. Thus, the behavioral data and the chemical analysis provide evidence that a specialized cuckoo wasp exhibits chemical mimicry of the odor of its host. This probably allows the cuckoo wasp to enter the nest with a reduced risk of being detected by olfaction and without leaving traitorous chemical traces.

Males of a solitary wasp possess a postpharyngeal gland.

The postpharyngeal gland has long been thought to occur only in ants. Here we characterize, by use of light and electron microscopy as well as 3D reconstruction based on nuclear magnetic resonance (NMR) imaging data, a large cephalic gland reservoir of males of a solitary digger wasp, the European beewolf, Philanthus triangulum. Several lines of evidence suggest that this reservoir is a postpharyngeal gland. The gland reservoir originates from the posterior part of the pharynx and consists of two pairs of unbranched tubular structures that occupy a large portion of the head capsule. Its wall is composed of a unicellular epithelium that is lined by a cuticle. The gland contains a blend of hydrocarbons and compounds with functional groups, and we show that the hydrocarbon fraction of the pheromone is congruent with the hydrocarbons on the cuticle. We discuss the implications of our findings for the evolution of the postpharyngeal gland in ants.

The odor of origin: kinship and geographical distance are reflected in the marking pheromone of male beewolves (Philanthus triangulum F., Hymenoptera, Crabronidae).

BACKGROUND: Pheromones play an important role for mate finding and courtship in many insects. In species where males are the signaling sex, females are expected to choose among potential mates with regard to the emitter's quality and/or genetic compatibility. One important aspect is the balance between negative and positive effects of in- vs. outbreeding. In the present study, we aimed to assess the potential of the territory marking pheromone of European beewolves as an indicator for genetic compatibility in the context of female choice. RESULTS: We analyzed the sex pheromone composition of male European beewolves (Philanthus triangulum F., Hymenoptera, Crabronidae) from eight different locations across Central Europe (six in Germany, one in England, and one in Italy). The pheromone constitutes a complex blend of various long-chain hydrocarbons (alkanes, alkenes, alcohols, ketones, and a carbon acid). We demonstrate that pheromone composition differs significantly among distant populations (regional scale), among subpopulations (local scale) and between families within subpopulations. The differences in the pheromone blend are positively correlated with geographical distances as might be expected according to an isolation-by-distance model. On a local scale, family membership has a larger effect on pheromone composition than subpopulation affiliation, while the reverse is true for the regional scale. CONCLUSION: Our results show that male pheromones can contain information on both kinship and geographical origin that may be used by females to choose adaptively among potential mates on the basis of their genetic distance.

A selfish function of a "social" gland? A postpharyngeal gland functions as a sex pheromone reservoir in males of the solitary wasp Philanthus triangulum.

The postpharyngeal gland (PPG) has long been assumed to be restricted to ants, where it mainly functions in the maintenance of social integrity. Recently, a PPG has been described in both sexes of a solitary digger wasp, the European beewolf, Philanthus triangulum (Hymenoptera, Crabronidae). Female beewolves use the contents of their PPG to embalm their honeybee prey to delay microbial growth. Here we show that in male beewolves, the PPG serves as a reservoir of the pheromone used to scent-mark their territories. Gas chromatography-mass spectrometry (GC-MS) analysis of PPG contents identified 55 substances including long-chain aliphatic hydrocarbons, and 13 substances with functional groups. The composition was consistent with the composition of the marking pheromone of male European beewolves described earlier. Comparisons of the PPG contents, and total-head extracts showed a strong congruency, suggesting that total-head extracts can be used for the analysis of marking secretion in beewolves. Furthermore, we found a dimorphism in the composition of the PPG contents, based on significant differences in the proportions of seven compounds between the two morphs.