The Chemistry of the Defensive Secretions of Three Species of Millipedes in the Genus Brachycybe.

Millipedes have long been known to produce a diverse array of chemical defense agents that deter predation. These compounds, or their precursors, are stored in high concentration within glands (ozadenes) and are released upon disturbance. The subterclass Colobognatha contains four orders of millipedes, all of which are known to produce terpenoid alkaloids-spare the Siphonophorida that produce terpenes. Although these compounds represent some of the most structurally-intriguing millipede-derived natural products, they are the least studied class of millipede defensive secretions. Here, we describe the chemistry of millipede defensive secretions from three species of Brachycybe: Brachycybe producta, Brachycybe petasata, and Brachycybe rosea. Chemical investigations using mass spectrometry-based metabolomics, chemical synthesis, and 2D NMR led to the identification of five alkaloids, three of which are new to the literature. All identified compounds are monoterpene alkaloids with the new compounds representing indolizidine (i.e. hydrogosodesmine) and quinolizidine alkaloids (i.e. homogosodesmine and homo-hydrogosodesmine). The chemical diversity of these compounds tracks the known species phylogeny of this genus, rather than the geographical proximity of the species. The indolizidines and quinolizidines are produced by non-sympatric sister species, B. producta and B. petasata, while deoxybuzonamine is produced by another set of non-sympatric sister species, B. rosea and Brachycybe lecontii. The fidelity between the chemical diversity and phylogeny strongly suggests that millipedes generate these complex defensive agents de novo and begins to provide insights into the evolution of their biochemical pathways.

Branched tyramides from males of the harvester ant, Pogonomyrmex badius.

Tyramides are produced in microgram quantities by males of species in the large Myrmicine ant sub-family (> 7000 species). Tyramides are transferred to female sexuals during mating where a specific female sexual evolved enzyme hydrolyzes the tyramides to the biogenic amine, tyramine. Tyramine is a ligand for receptors that rapidly activate reproductive development in the newly mated queen-previously reproductively inhibited by the mother queen. Without this elaborate biogenic amine precursor and co-evolved female sexual derived tyramide hydrolase, the defenseless newly mated queen's worker production would be delayed by up to 6 days, which could be lethal to the new queen. This is one of possibly several ant species separation mechanisms evolved to maintain species integrity. Here we report two methyl-branched tyramides from harvester ant, Pogonomyrmex badius, males, including one highly branched tyramide not previously reported.

Novel alkaloids from the fire ant, Solenopsis geminata.

South American fire ants, Solenopsis richteri and Solenopsis invicta, were accidently introduced into the southern USA in the 1900s and 1930s, respectively. The rapid spread and high population densities of S. invicta, and its potent sting, resulted in broad economic impacts and a variety of research efforts. In the 1970s, their venom alkaloids were identified as a complex blend of trans-2-methyl-6-alkyl- and alkenyl-piperidines. Solenopsis geminata is a worldwide tramp species but a native of the southern coastal regions of the USA. It was found to only produce cis- and trans-2-methyl-6-undecyl-piperidines. These alkaloids were considered the Solenopsis ancestral alkaloid profile since they were identified from female sexuals (potential queens) of all Solenopsis species in South and North America. The dramatic modification of alkaloids in Solenopsis invicta was attributed to their response to evolutionary pressure and the lack of change in S. geminata alkaloids due to no response to evolutionary pressure. Here we report the unexpected discovery of 6-undecyl-pyridine, 2-methyl-6-undecyl-pyridine and 2-methyl-6-(1)-undecenyl-pyridine as components of S. geminata worker venom, suggesting that S. geminata like its South American relatives have responded to evolutionary pressures. Our results will stimulate future research on S. geminata populations throughout the tropical/subtropical world.

The Evolution of Tyramides in Male Fungus-Growing Ants (Formicidae: Myrmicinae: Attini: Attina).

Ants use a variety of semiochemicals for essential activities and have been a source for many novel natural products. While ant taxa produce a wide variety of chemicals, the chemistry and ecology of male ants have remained understudied. Tyramides are a class of compounds that have been found only in males of the Myrmicinae ant subfamily. Tyramides found in the fire ant Solenopsis invicta are transferred to gynes during mating where they are converted to tyramine, leading to rapid reproductive development. To further understand the evolution of tyramide production in male ants, we determined the tyramide composition in males of 15 fungus-growing ant species (Formicidae: Myrmicinae: Attini: Attina) and a Megalomyrmex species (Formicidae: Myrmicinae: Solenopsidini). Thirteen tyramides were identified, four for the first time in natural sources, and their percent composition was mapped to the fungus-growing ant phylogeny.

Deoxybuzonamine Isomers from the Millipede Brachycybe lecontii (Platydesmida: Andrognathidae).

Millipedes (Diplopoda) are well known for their toxic or repellent defensive secretions. Here, we describe (6aR,10aS,10bR)-8,8-dimethyldodecahydropyrrolo[2,1-a]isoquinoline [trans-anti-trans-deoxybuzonamine (1a)] and (rel-6aR,10aR,10bR)-8,8-dimethyldodecahydropyrrolo[2,1-a]isoquinoline [trans-syn-cis-deoxybuzonamine (1b)], two isomers of deoxybuzonamine found in the chemical defense secretions of the millipede Brachycybe lecontii Wood (Colobognatha, Platydesmida, Andrognathidae). The carbon-nitrogen skeleton of these compounds was determined from their MS and GC-FTIR spectra obtained from the MeOH extract of whole millipedes, along with a subsequent selective synthesis. Their structures were established from their 1D (1H, 13C) and 2D NMR (COSY, NOESY, multiplicity-edited HSQC, HSQC-TOCSY, HMBC) spectra. Additionally, computational chemistry (DFT and DP4) was used to identify the relative configurations of 1a and 1b by comparing predicted 13C data to their experimental values, and the absolute configuration of 1a was determined by comparing its experimental specific rotation with that of the computationally calculated value. This is the first report of dodecahydropyrrolo[2,1-a]isoquinoline alkaloids from a platydesmidan millipede.

Gosodesmine, a 7-Substituted Hexahydroindolizine from the Millipede Gosodesmus claremontus.

Millipedes (Diplopoda) are well known for their toxic or repellent defensive secretions. Here we describe gosodesmine (1), 7-(4-methylpent-3-en-1-yl)-1,2,3,5,8,8a-hexahydroindolizine, a unique alkaloid with some terpene character found in the chemical defense secretions of the millipede Gosodesmus claremontus Chamberlin (Colobognatha, Platydesmida, Andrognathidae). The structure of 1 was suggested by its mass spectra and GC-FTIR spectra and established from its 1H, 13C, and 2D NMR spectra and 1D NOE studies. The 7-substituted indolizidine carbon skeleton of 1 was confirmed by unambiguous synthesis. This is the first report of an alkaloid from a platydesmid millipede and the first report of a 7-substituted indolizidine from an arthropod.

The Chemistry of Some Dalodesmidean Millipedes from Tasmania (Diplopoda, Polydesmida).

Millipedes (Diplopoda) are well known for their toxic or repellent defensive secretions. As part of a larger investigation, we describe the chemical constituents of 14 species of Tasmanian millipedes in seven genera. Six species in the genus Gasterogramma were found to produce acyclic ketones, including the pungent unsaturated ketones 1, 2, and 6, and the novel (rel-3R,5S,7S)-3,5,7-trimethyl-2,8-decanedione (7b), for which the stereoconfiguration was established by stereoselective syntheses of pairs of isomers. These compounds have not been detected before in millipede defensive secretions. This report is the first on species of the suborder Dalodesmidea (Polydesmida), a dominant component of the soil and litter fauna of the temperate regions of the Southern Hemisphere.

Chemically armed mercenary ants protect fungus-farming societies.

The ants are extraordinary in having evolved many lineages that exploit closely related ant societies as social parasites, but social parasitism by distantly related ants is rare. Here we document the interaction dynamics among a Sericomyrmex fungus-growing ant host, a permanently associated parasitic guest ant of the genus Megalomyrmex, and a raiding agro-predator of the genus Gnamptogenys. We show experimentally that the guest ants protect their host colonies against agro-predator raids using alkaloid venom that is much more potent than the biting defenses of the host ants. Relatively few guest ants are sufficient to kill raiders that invariably exterminate host nests without a cohabiting guest ant colony. We also show that the odor of guest ants discourages raider scouts from recruiting nestmates to host colonies. Our results imply that Sericomyrmex fungus-growers obtain a net benefit from their costly guest ants behaving as a functional soldier caste to meet lethal threats from agro-predator raiders. The fundamentally different life histories of the agro-predators and guest ants appear to facilitate their coexistence in a negative frequency-dependent manner. Because a guest ant colony is committed for life to a single host colony, the guests would harm their own interests by not defending the host that they continue to exploit. This conditional mutualism is analogous to chronic sickle cell anemia enhancing the resistance to malaria and to episodes in human history when mercenary city defenders offered either net benefits or imposed net costs, depending on the level of threat from invading armies.

Alkaloid venom weaponry of three Megalomyrmex thief ants and the behavioral response of Cyphomyrmex costatus host ants.

Social parasites exploit other societies by invading and stealing resources. Some enter protected nests using offensive chemical weaponry made from alkaloid-based venom. We characterized the venoms of three Megalomyrmex thief ant species (M. mondabora, M. mondaboroides, and M. silvestrii) that parasitize the fungus-growing ants, and developed an ethogram to describe host ant reactions to raiding M. mondaboroides and M. silvestrii parasites. We compared piperidine, pyrrolidine, and pyrolizidine venom alkaloid structures with synthetic samples from previous studies, and describe the novel stereochemistry of trans 2-hexyl-5-[8-oxononyl]-pyrrolidine (3) from M. mondabora. We showed that workers of Cyphomyrmex costatus, the host of M. mondaboroides and M. silvestrii, react to a sting by Megalomyrmex parasites mainly with submissive behavior, playing dead or retreating. Host submission also followed brief antennal contact. The behavior of C. costatus ants observed in this study was similar to that of Cyphomyrmex cornutus, host of M. mondabora, suggesting that the alkaloidal venoms with pyrrolidines from M. mondabora, piperidines from M. mondaboroides, and pyrolizidines from M. silvestrii may function similarly as appeasement and repellent allomones against host ants, despite their different chemical structure. With the use of these chemical weapons, the Megalomyrmex thief ants are met with little host resistance and easily exploit host colony resources.

Characterization of physical and chemical defenses in the hemlock woolly adelgid.

The invasive hemlock woolly adelgid (Adelges tsugae Annand, Hemiptera: Sternorrhyncha: Adelgidae) causes significant mortality to eastern and Carolina hemlock (Tsuga canadensis Carrière and T. caroliniana Engelmann, respectively) throughout the eastern United States. Adelges tsugae produces vast quantities of a wax covering that surrounds most of the instars as well as the adult and eggs. Using direct probe EI mass spectrometry, this wax covering was characterized as a diketoester wax, 17-oxohexatriacontanyl 11-oxotriacontanoate, and accounted for ≈ 42 % of A. tsugae total biomass. The presence of the anthraquinone, chrysophanol, and its precursor anthrone, chrysarobin, in A. tsugae has only been briefly described. Further study confirmed these compounds in all A. tsugae life stages. Additionally, several predatory beetles in use or under consideration as biological control agents for this species appear to ingest and excrete these compounds when feeding on A. tsugae. The production of both a physical and a chemical defense may represent a significant energy investment by A. tsugae.

Investigating Photo-Degradation as a Potential Pheromone Production Pathway in Spotted Lanternfly, Lycorma delicatula.

Since its discovery in North America in 2014, the spotted lanternfly (SLF), Lycorma delicatula, has become an economic, ecological, and nuisance pest there. Developing early detection and monitoring tools is critical to their mitigation and control. Previous research found evidence that SLF may use pheromones to help locate each other for aggregation or mating. Pheromone production necessitates specific conditions by the insects, and these must be investigated and described. A chemical process called photo-degradation has been described as a final step in the production of pheromones in several diurnal insect species, in which cuticular hydrocarbons were broken down by sunlight into volatile pheromone components. In this study, photo-degradation was investigated as a possible pheromone production pathway for SLF. Extracts from SLF mixed-sex third and fourth nymphs and male or female adults were either exposed to simulated sunlight to produce a photo-degradative reaction (photo-degraded), or not exposed to light (crude), while volatiles were collected. Behavioral bioassays tested for attraction to volatiles from photo-degraded and crude samples and their residues. In third instars, only the volatile samples from photo-degraded mixed-sex extracts were attractive. Fourth instar males were attracted to both crude and photo-degraded residues, and volatiles of photo-degraded mixed-sex extracts. Fourth instar females were attracted to volatiles of crude and photo-degraded mixed-sex extracts, but not to residues. In adults, only males were attracted to body volatiles from crude and photo-degraded extracts of either sex. Examination of all volatile samples using gas chromatography coupled with mass spectrometry (GC-MS) revealed that most of the identified compounds in photo-degraded extracts were also present in crude extracts. However, the abundance of these compounds in photo-degraded samples were 10 to 250 times more than their abundance in the crude counterparts. Results from behavioral bioassays indicate that photo-degradation probably does not generate a long-range pheromone, but it may be involved in the production of a short-range sex-recognition pheromone in SLF. This study provides additional evidence of pheromonal activity in SLF.

Potential feeding deterrents found in hemlock woolly adelgid, Adelges tsugae.

The nonnative hemlock woolly adelgid (Adelges tsugae Annand, Hemiptera: Sternorrhyncha: Adelgidae) has been a significant mortality agent of eastern hemlock (Tsuga canadensis Carriere) throughout a large portion of its geographic range. During a study investigating adelgid vigor in relation to host health, it was noted that adelgid extracts ranged from a yellow to a deep red color. Analysis by GC-MS identified the presence of the anthraquinone, chrysophanol and its anthrone precursor, chrysarobin in the extract. These compounds are predator deterrents in several other insects, including chrysomelid beetles. It is hypothesized that these compounds serve a similar purpose in the hemlock woolly adelgid.

Histrionicotoxin alkaloids finally detected in an ant.

Workers of the ant Carebarella bicolor collected in Panama were found to have two major poison-frog alkaloids, cis- and trans-fused decahydroquinolines (DHQs) of the 269AB type, four minor 269AB isomers, two minor 269B isomers, and three isomers of DHQ 271D. For the first time in an ant, however, the DHQs were accompanied by six histrionicotoxins (HTXs), viz., 283A, 285A, 285B, 285C, 287A, and 287D. This co-occurrence of the HTX and DHQ alkaloids is the usual pattern seen in dendrobatid frogs. This finding contrasts with our earlier study, where workers of a Brazilian ant, Solenopsis (Diplorhoptrum) sp., were found to have a very similar DHQ complex but failed to show HTXs. Several new DHQ alkaloids of MW 271 (named in the frog as 271G) are reported from the above ants that have both m/z 202 and 204 as major fragment ions, unlike the spectrum seen for the poison-frog alkaloid 271D, which has only an m/z 204 base peak. Found also for the first time in skin extracts from the comparison frog Oophaga granulifera of Costa Rica is a trace DHQ of MW 273. It is coded as 273F in the frog; a different isomer is found in the ant.

Male-produced pheromone in the European woodwasp, Sirex noctilio.

A male-produced pheromone that attracts both males and females was identified for the European woodwasp, Sirex noctilio, a serious pest of pine trees. Males displayed excitatory behaviors when placed in groups, and were attracted to the odors from males that were 2-5-d-old, but not to odors from males that were 0-1-d-old. An unsaturated short-chain alcohol, (Z)-3-decen-1-ol, was discovered in samples collected on SuperQ filters over groups of males and identified by using micro-derivatization reactions and gas chromatography coupled with mass spectrometry (GC-MS). The compound was not detected in volatile samples from females. Gas chromatography coupled electroantennographic detection (GC-EAD) of antennae from males exposed to male headspace odors produced strong antennal responses to the main peak of (Z)-3-decen-1-ol, as well as to an unknown minor component that had a similar retention time. Antennae from both males and females responded to synthetic (Z)-3-decen-1-ol. Several different synthetic candidates for the GC-EAD active minor components were selected based on GC-MS and GC-EAD responses to male headspace collections. These synthetic compounds were tested for antennal activity using GC-EAD, and those that produced strong responses were blended with the major component and tested for male attraction in the Y-tube olfactometer at different concentrations and ratios. Males tested in the Y-tube olfactometer were attracted to a synthetic blend of (Z)-3-decen-1-ol and (Z)-4-decen-1-ol at a ratio of 100:1. Whereas the addition of some suspected minor compounds reduced attraction, the addition of a third compound found in male emanations that produced strong male antennal responses, (E,E)-2,4-decadienal (at a ratio of 100:1:1), resulted in attraction of both males (Y-tube and wind tunnel) and females (wind tunnel).

Using an integrative taxonomic approach to delimit a sibling species, Mycetomoellerius mikromelanos sp. nov. (Formicidae: Attini: Attina).

The fungus-growing ant Mycetomoellerius (previously Trachymyrmex) zeteki (Weber 1940) has been the focus of a wide range of studies examining symbiotic partners, garden pathogens, mating frequencies, and genomics. This is in part due to the ease of collecting colonies from creek embankments and its high abundance in the Panama Canal region. The original description was based on samples collected on Barro Colorado Island (BCI), Panama. However, most subsequent studies have sampled populations on the mainland 15 km southeast of BCI. Herein we show that two sibling ant species live in sympatry on the mainland: Mycetomoellerius mikromelanos Cardenas, Schultz, & Adams and M. zeteki. This distinction was originally based on behavioral differences of workers in the field and on queen morphology (M. mikromelanos workers and queens are smaller and black while those of M. zeteki are larger and red). Authors frequently refer to either species as "M. cf. zeteki," indicating uncertainty about identity. We used an integrative taxonomic approach to resolve this, examining worker behavior, chemical profiles of worker volatiles, molecular markers, and morphology of all castes. For the latter, we used conventional taxonomic indicators from nine measurements, six extrapolated indices, and morphological characters. We document a new observation of a Diapriinae (Hymenoptera: Diapriidae) parasitoid wasp parasitizing M. zeteki. Finally, we discuss the importance of vouchering in dependable, accessible museum collections and provide a table of previously published papers to clarify the usage of the name T. zeteki. We found that most reports of M. zeteki or M. cf. zeteki-including a genome-actually refer to the new species M. mikromelanos.

Male fire ant neurotransmitter precursors trigger reproductive development in females after mating.

Social insect queens have evolved mechanisms to prevent competition from their sexual daughters. For Solenopsis invicta, the fire ant, queens have evolved a primer pheromone that retards reproductive development in their winged reproductive daughters. If these daughters are removed from the influence of the queen, it takes about a week to start reproductive development; however, it starts almost immediately after mating. This dichotomy has been unsuccessfully investigated for several decades. Here we show that male fire ants produce tyramides, derivatives of the biogenic amine tyramine, in their reproductive system. Males transfer tyramides to winged females during mating, where the now newly mated queens enzymatically convert tyramides to tyramine. Tyramine floods the hemolymph, rapidly activating physiological processes associated with reproductive development. Tyramides have been found only in the large Myrmicinae ant sub-family (6,800 species), We suggest that the complex inhibition/disinhibition of reproductive development described here will be applicable to other members of this ant sub-family.

Caste-specific tyramides from Myrmicine ants.

Analysis of the extracts of male ants of Monomorium minimum and Monomorium ebeninum by GC-MS and GC-FTIR revealed the presence of tyramides 2 and 4c, for which the structures were established by comparison with synthetic samples. These compounds and their analogues 1 and 3 were also found in males of other Monomorium species, males of Myrmicaria opaciventris, and males of several Solenopsis (Diplorhoptrum) species. Vapor-phase FTIR spectra revealed critically important structural clues to two of the tyramides, which had methyl branching in the tyramide acyl moiety. Tyramide 4c exhibited a strong intramolecular amide NH hydrogen bond where an alpha-keto group was deduced to be present in the acyl moiety and also showed the overlap of this ketone group frequency with that of the amide nu(C horizontal lineO). The biological function of these compounds is uncertain; however, their role in ant-mating behavior may be suggested by a large body of evidence.

Venom Function of a New Species of Megalomyrmex Forel, 1885 (Hymenoptera: Formicidae).

Alkaloids are important metabolites found across a variety of organisms with diverse ecological functions. Of particular interest are alkaloids found in ants, organisms well known for dominating the ecosystems they dwell in. Within ants, alkaloids are found in venom and function as potent weapons against heterospecific species. However, research is often limited to pest species or species with parasitic lifestyles and thus fails to address the broader ecological function of ant venom alkaloids. Here we describe a new species of free-living Megalomyrmex ant: Megalomyrmex peetersi sp. n. In addition, we identify its singular venom alkaloid (trans-2-butyl-5-heptylpyrrolidine) and elucidate the antibiotic and insecticidal functions of its venom. Our results show that Megalomyrmex peetersi sp. n. venom is an effective antibiotic and insecticide. These results are comparable to venom alkaloids found in other ant species, such as Solenopsis invicta. This research provides great insight into venom alkaloid function, and it is the first study to explore these ideas in the Megalomyrmex system.

Behavioral evidence for a contact sex pheromone component of the emerald ash borer, Agrilus planipennis Fairmaire.

The cuticular hydrocarbon profiles of emerald ash borers, Agrilus planipennis, were examined to determine if there are differences in these compounds between the sexes. We also assessed feral male EAB in the field for behavioral changes based on the application of a female-specific compound to dead, solvent-washed beetles. Males in the field spent significantly more time attempting copulation with dead, pinned female beetles coated with a three-beetle-equivalent dose of 3-methyltricosane than with solvent-washed beetles or those coated in 3-methyltricosane at lower concentrations. Males in the field spent the most time investigating pinned dead, unwashed female beetles. In the laboratory, sexually mature males were presented with one of several mixtures applied in hexane to filter paper disks or to the elytra of dead female beetles first washed in solvent. Male EAB also spent more time investigating dead beetles treated with solution applications that contained 3-methyltricosane than dead beetles and filter paper disks treated with male body wash or a straight-chain hydrocarbon not found on the cuticle of EAB.

Monoalkenes as contact sex pheromone components of the woodwasp Sirex noctilio.

A pheromone on the cuticle of females of the woodwasp Sirex noctilio, a recently introduced pest of pines in North America, induces conspecific males to attempt copulation. Dead females washed with hexane did not elicit copulation attempts from males, whereas reapplication of a female hexane body wash onto the cuticle of dead females elicited copulation attempts by 65% of males tested. Analysis of the hexane extract revealed saturated and unsaturated hydrocarbons as major components of the female cuticle. Behavior-guided fractionation of the female body wash led to the identification of three components, (Z)-7-heptacosene, (Z)-7-nonacosene, and (Z)-9-nonacosene, of the sex pheromone of S. noctilio that elicited copulatory responses from males.