Cardenolides, toxicity, and the costs of sequestration in the coevolutionary interaction between monarchs and milkweeds.

For highly specialized insect herbivores, plant chemical defenses are often co-opted as cues for oviposition and sequestration. In such interactions, can plants evolve novel defenses, pushing herbivores to trade off benefits of specialization with costs of coping with toxins? We tested how variation in milkweed toxins (cardenolides) impacted monarch butterfly (Danaus plexippus) growth, sequestration, and oviposition when consuming tropical milkweed (Asclepias curassavica), one of two critical host plants worldwide. The most abundant leaf toxin, highly apolar and thiazolidine ring-containing voruscharin, accounted for 40% of leaf cardenolides, negatively predicted caterpillar growth, and was not sequestered. Using whole plants and purified voruscharin, we show that monarch caterpillars convert voruscharin to calotropin and calactin in vivo, imposing a burden on growth. As shown by in vitro experiments, this conversion is facilitated by temperature and alkaline pH. We next employed toxin-target site experiments with isolated cardenolides and the monarch's neural Na+/K+-ATPase, revealing that voruscharin is highly inhibitory compared with several standards and sequestered cardenolides. The monarch's typical >50-fold enhanced resistance to cardenolides compared with sensitive animals was absent for voruscharin, suggesting highly specific plant defense. Finally, oviposition was greatest on intermediate cardenolide plants, supporting the notion of a trade-off between benefits and costs of sequestration for this highly specialized herbivore. There is apparently ample opportunity for continued coevolution between monarchs and milkweeds, although the diffuse nature of the interaction, due to migration and interaction with multiple milkweeds, may limit the ability of monarchs to counteradapt.

Cuticular and Dufour's Gland Chemistry Reflect Reproductive and Social State in the Facultatively Eusocial Sweat Bee Megalopta genalis (Hymenoptera: Halictidae).

Queen pheromones evolved independently in multiple eusocial insect lineages, in which they mediate reproductive conflict by inhibiting worker ovarian development. Although fundamentally important for reproductive division of labor - the hallmark of eusociality - their evolutionary origins are enigmatic. Here, we analyze cuticular and Dufour's gland chemistries across alternative social and reproductive phenotypes in Megalopta genalis bees (tribe Augochlorini, family Halictidae) that facultatively express simple eusociality. Reproductive bees have distinct overall glandular and cuticular chemical phenotypes compared with non-reproductive workers. On the cuticle, a likely site of signal transmission, reproductives are enriched for certain alkenes, most linear alkanes, and are heavily enriched for all methyl-branched alkanes. Chemicals belonging to these compound classes are known to function as fertility signals in other eusocial insect taxa. Some macrocyclic lactones, compounds that serve as queen pheromones in the other eusocial halictid tribe (Halictini), are also enriched among reproductives relative to workers. The intra-population facultative eusociality of M. genalis permits direct comparisons between individuals expressing alternative reproductive phenotypes - females that reproduce alone (solitary reproductives) and social queens - to highlight traits in the latter that may be important mediators of eusociality. Compared with solitary reproductives, the cuticular chemistries of queens are more strongly differentiated from those of workers, and furthermore are especially enriched for methyl-branched alkanes. Determining the pheromonal function(s) and information content of the candidate signaling compounds we identify will help illuminate the early evolutionary history of queen pheromones, chemical signals central to the organization of insect eusocial behavior.

Ecological fitting: Chemical profiles of plant hosts provide insights on selection cues and preferences for a major buprestid pest.

Specific cues used by emerald ash borer (EAB, Agrilus planipennis) to select hosts are largely unknown. Attractants are likely general and the use of novel host plants provides an opportunity to investigate the commonality of these cues. We examined volatile profiles emitted by five plants that can host EAB and estimated their importance in explaining known oviposition preferences. Foliage volatiles were collected from potted black ash (Fraxinus nigra), Manchurian ash (F. mandshurica), blue ash (F. quadrangulata), white fringetree (Chionanthus virginicus), and olive (Olea europaea) and analyzed using GC-MS. Fifty-nine compounds were detected including eight green leaf volatiles (GLV), 12 monoterpenes, and 21 sesquiterpenes. Ordination plots show separation of species by full foliage profiles, monoterpenes, sesquiterpenes, and known antennally active compounds, but GLVs were similar across hosts. Random Forest (RF) analysis revealed eight compounds that separated plant species with an error rate of ~19%, consisting mostly of sesquiterpenes. Similarity of GLV profiles among known hosts suggests they serve as general cues for host selection. Manchurian ash, a resistant host, produced the highest quantities and variety of sesquiterpenes indicating that some of these chemicals may be antixenotic. All compounds identified by RF have been implicated as deterrents or attractants to woodborers in other studies and should be investigated for adult antennal activity and attraction.

Identification of a queen and king recognition pheromone in the subterranean termite Reticulitermes flavipes.

Chemical communication is fundamental to success in social insect colonies. Species-, colony-, and caste-specific blends of cuticular hydrocarbons (CHCs) and other chemicals have been well documented as pheromones, mediating important behavioral and physiological aspects of social insects. More specifically, royal pheromones used by queens (and kings in termites) enable workers to recognize and care for these vital individuals and maintain the reproductive division of labor. In termites, however, no royal-recognition pheromones have been identified to date. In the current study, solvent extracts of the subterranean termite Reticulitermes flavipes were analyzed to assess differences in cuticular compounds among castes. We identified a royal-specific hydrocarbon-heneicosane-and several previously unreported and highly royal enriched long-chain alkanes. When applied to glass dummies, heneicosane elicited worker behavioral responses identical to those elicited by live termite queens, including increased vibratory shaking and antennation. Further, the behavioral effects of heneicosane were amplified when presented with nestmate termite workers' cuticular extracts, underscoring the importance of chemical context in termite royal recognition. Thus, heneicosane is a royal-recognition pheromone that is active in both queens and kings of R. flavipes The use of heneicosane as a queen and king recognition pheromone by termites suggests that CHCs evolved as royal pheromones ∼150 million years ago, ∼50 million years before their first use as queen-recognition pheromones in social Hymenoptera. We therefore infer that termites and social Hymenoptera convergently evolved the use of these ubiquitous compounds in royal recognition.

High pesticide risk to honey bees despite low focal crop pollen collection during pollination of a mass blooming crop.

Honey bees provide critical pollination services for many agricultural crops. While the contribution of pesticides to current hive loss rates is debated, remarkably little is known regarding the magnitude of risk to bees and mechanisms of exposure during pollination. Here, we show that pesticide risk in recently accumulated beebread was above regulatory agency levels of concern for acute or chronic exposure at 5 and 22 of the 30 apple orchards, respectively, where we placed 120 experimental hives. Landscape context strongly predicted focal crop pollen foraging and total pesticide residues, which were dominated by fungicides. Yet focal crop pollen foraging was a poor predictor of pesticide risk, which was driven primarily by insecticides. Instead, risk was positively related to diversity of non-focal crop pollen sources. Furthermore, over 60% of pesticide risk was attributed to pesticides that were not sprayed during the apple bloom period. These results suggest the majority of pesticide risk to honey bees providing pollination services came from residues in non-focal crop pollen, likely contaminated wildflowers or other sources. We suggest a greater understanding of the specific mechanisms of non-focal crop pesticide exposure is essential for minimizing risk to bees and improving the sustainability of grower pest management programs.

Honey Bee (Apis mellifera) Queen Reproductive Potential Affects Queen Mandibular Gland Pheromone Composition and Worker Retinue Response.

Reproductive division of labor is one of the defining traits of honey bees (Apis mellifera), with non-reproductive tasks being performed by workers while a single queen normally monopolizes reproduction. The decentralized organization of a honey bee colony is maintained in large part by a bouquet of queen-produced pheromones, the distribution of which is facilitated by contact among workers throughout the hive. Previous studies have shown that the developmental fate of honey bee queens is highly plastic, with queens raised from younger worker larvae exhibiting higher measures of reproductive potential compared to queens raised from older worker larvae. We investigated differences in the chemical composition of the mandibular glands and attractiveness to workers of "high-quality" queens (i.e., raised from first instar worker larvae; more queen-like) and "low-quality" queens (i.e., raised from third instar worker larvae; more worker-like). We characterized the chemical profiles of the mandibular glands of high-quality queens and low-quality queens using GC-MS and used the worker retinue response as a measure of the attractiveness to workers of high-quality queens vs. low-quality queens. We found that queen quality affected the chemical profiles of mandibular gland contents differently across years, showing significant differences in the production of the queen mandibular pheromone ("QMP") components HVA and 9-HDA in 2010, but no significant differences of any glandular compound in 2012. We also found that workers were significantly more attracted to high-quality queens than to low-quality queens in 2012, possibly because of increased attractiveness of their mandibular gland chemical profiles. Our results indicate that the age at which honey bee larvae enter the "queen-specific" developmental pathway influences the chemical composition of queen mandibular glands and worker behavior. However, these changes are not consistent across years, suggesting that other external factors may play important roles in modulating queen quality.

Genetic architecture of natural variation in cuticular hydrocarbon composition in Drosophila melanogaster.

Insect cuticular hydrocarbons (CHCs) prevent desiccation and serve as chemical signals that mediate social interactions. Drosophila melanogaster CHCs have been studied extensively, but the genetic basis for individual variation in CHC composition is largely unknown. We quantified variation in CHC profiles in the D. melanogaster Genetic Reference Panel (DGRP) and identified novel CHCs. We used principal component (PC) analysis to extract PCs that explain the majority of CHC variation and identified polymorphisms in or near 305 and 173 genes in females and males, respectively, associated with variation in these PCs. In addition, 17 DGRP lines contain the functional Desat2 allele characteristic of African and Caribbean D. melanogaster females (more 5,9-C27:2 and less 7,11-C27:2, female sex pheromone isomers). Disruption of expression of 24 candidate genes affected CHC composition in at least one sex. These genes are associated with fatty acid metabolism and represent mechanistic targets for individual variation in CHC composition.

Insects groom their antennae to enhance olfactory acuity.

Grooming, a common behavior in animals, serves the important function of removing foreign materials from body surfaces. When antennal grooming was prevented in the American cockroach, Periplaneta americana, field emission gun scanning electron microscopy images revealed that an unstructured substance accumulated on nongroomed antennae, covering sensillar pores, but not on groomed antennae of the same individuals. Gas chromatography analysis of antennal extracts showed that over a 24-h period nongroomed antennae accumulated three to four times more cuticular hydrocarbons than groomed antennae. Moreover, nongroomed antennae accumulated significantly more environmental contaminants from surfaces (stearic acid) and from air (geranyl acetate) than groomed antennae. We hypothesized that the accumulation of excess native cuticular hydrocarbons on the antennae would impair olfactory reception. Electroantennogram experiments and single-sensillum recordings supported this hypothesis: antennae that were prevented from being groomed were significantly less responsive than groomed antennae to the sex pheromone component periplanone-B, as well as to the general odorants geranyl acetate and hexanol. We therefore conclude that antennal grooming removes excess native cuticular lipids and foreign chemicals that physically and/or chemically interfere with olfaction, and thus maintains the olfactory acuity of the antennae. Similar experimental manipulations of the German cockroach (Blattella germanica), carpenter ant (Camponotus pennsylvanicus), and the housefly (Musca domestica), which use different modes of antennal grooming, support the hypothesis that antennal grooming serves a similar function in a wide range of insect taxa.

Volatile profile differences and the associated Sirex noctilio activity in two host tree species in the Northeastern United States.

Sirex noctilio females are known to be attracted to stem sections of stressed pine trees for oviposition. The volatile profiles and attractiveness of Eastern white pine (Pinus strobus) and two chemotypes of Scots pine (P. sylvestris) were compared after stem injection with herbicide. In general, trap captures on herbicide-treated trees were higher than on controls. The high-carene chemotype of Scots pine captured the highest numbers of females, followed by the low-carene chemotype, and finally the Eastern white pine. Herbicide-treated trees of both species emitted larger quantities of volatiles than the controls. The herbicide treatment induced higher volatile emission rates in the Scots pine chemotypes than in white pine, although there was no difference between the two chemotypes. However, qualitative differences were found between the volatile profiles of the two species as well as between the two Scots pine chemotypes, which could account for the differential attractiveness of the species and chemotypes tested.

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).

Bacteria stimulate hatching of yellow fever mosquito eggs.

BACKGROUND: Aedes aegypti Linnaeus is a peridomestic mosquito that lays desiccation-resistant eggs in water-filled human-made containers. Previous investigations connected egg hatching with declining dissolved oxygen (DO) that is associated with bacterial growth. However, past studies failed to uncouple DO from other potential stimulatory factors and they contained little quantitative information about the microbial community; consequently, a direct role for bacteria or compounds associated with bacteria in stimulating egg hatching cannot be dismissed. METHODOLOGY/PRINCIPAL FINDINGS: Environmental factors stimulating hatch of Ae. aegypti eggs were investigated using non-sterile and sterile white oak leaf (WOL) infusions and a bacterial culture composed of a mix of 14 species originally isolated from bamboo leaf infusion. In WOL infusion with active microbes, 92.4% of eggs hatched in 2-h at an average DO concentration of 2.4 ppm. A 24-h old bacterial culture with a DO concentration of 0.73 ppm also stimulated 95.2% of eggs hatch within 1-h. In contrast, only 4.0% of eggs hatched in sterile infusion, whose DO averaged 7.4 ppm. Effects of bacteria were uncoupled from DO by exposing eggs to bacterial cells suspended in NaCl solution. Over a 4-h exposure period, 93.8% of eggs hatched while DO concentration changed minimally from 7.62 to 7.50 ppm. Removal of bacteria by ultra-filtration and cell-free filtrate resulted in only 52.0% of eggs hatching after 4-h at an average DO concentration of 5.5 ppm. CONCLUSIONS/SIGNIFICANCE: Collectively, the results provide compelling evidence that bacteria or water-soluble compounds secreted by bacteria, not just low DO concentration, stimulate hatching of Ae. aegypti eggs. However, the specific cues involved remain to be identified. These research findings contribute new insight into an important aspect of the oviposition biology of Ae. aegypti, a virus vector of global importance, providing the basis for a new paradigm of environmental factors involved in egg hatching.

Pea aphids, Acyrthosiphon pisum, suppress induced plant volatiles in broad bean, Vicia faba.

Plants defend themselves against herbivory through several means, including the production of airborne volatile organic compounds (VOCs). These VOCs benefit plants by attracting natural enemies of their herbivores. The pea aphid, Acyrthosiphon pisum, is able to feed on its host plant, Vicia faba, without inducing detectable changes in plant VOC emission. Levels of VOCs emission are not significantly different between control plants and those fed upon by aphids for up to 5 days. Using a second herbivore, the beet armyworm caterpillar, Spodoptera exigua, we demonstrate that several expected caterpillar-induced VOCs are reduced when co-infested with pea aphids, thus demonstrating that pea aphids have the ability to inhibit the release of certain VOCs. This study shows, for the first time, that aphids not only avoid triggering plant volatile emission, but also can actively inhibit herbivore-induced volatiles.

Oviposition responses of the mosquitoes Aedes aegypti and Aedes albopictus to experimental plant infusions in laboratory bioassays.

Attraction of the mosquitoes Aedes aegypti and Ae. albopictus to plant infusions was evaluated by using a modified sticky-screen bioassay that improved the resolution of mosquito responses to odorants. Under bioassay conditions, solid-phase microextraction-gas chromatographic analyses of the volatile marker chemical indole showed that odorants diffused from bioassay cups, forming a concentration gradient. Infusions were prepared by separately fermenting senescent leaves of eight plant species in well water. Plant infusions were evaluated over an 8-fold range of leaf biomass and/or a 28 d fermentation period. The responses of gravid females of both mosquito species varied with the plant species and biomass of plant materials used to make infusions, and with the length of the fermentation period. Infusions made from senescent bamboo (Arundinaria gigantea) and white oak (Quercus alba) leaves were significantly attractive to both mosquitoes. In general, infusions prepared by using low biomass of plant material over a 7-14 d fermentation period were most attractive to Ae. aegypti. In contrast, Ae. albopictus was attracted to infusions made using a wider range of plant biomass and over a longer fermentation period. Both mosquito species were more attracted to a non-sterile white oak leaf infusion than to white oak leaf infusion that was prepared using sterilized plant material and water, thus suggesting a role for microbial activity in the production of odorants that mediate the oviposition response of gravid mosquitoes.

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.

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.

Differences in cuticular lipid composition of the antennae of Helicoverpa zea, Heliothis virescens, and Manduca sexta.

Analyses of the hexane washes of antennae, forelegs and whole bodies of Helicoverpa zea, Heliothis virescens, and Manduca sexta revealed notable differences in the components of the cuticular coatings of each species. Most striking were the differences between the cuticular coatings of male and female antennae of both H. zea and H. virescens. Novel esters of short-chain acids (C2-C4) and long-chain secondary alcohols (C25-C32) were identified in the hexane washes of the male antenna and forelegs of H. zea and H. virescens. These compounds were found in only small amounts or were completely absent on the female antennae of both species. In H. zea, butyrates of 7- and 8-pentacosanol and 8- and 9-heptacosanol were found, whereas, in the foreleg extracts of H. virescens, acetates and propionates were detected in addition to butyrates. While cholesterol is a major component of antennal washes (10-15%), only traces were found in the foreleg extracts. Although the composition of the cuticular coating of M. sexta differed greatly from that of the other two species, the extractable coatings of the antennae of male and female M. sexta were nearly identical.

Identification, synthesis, and conformation of tri- and tetrathiacycloalkanes from marine bacteria.

Seven new cyclic natural polysulfides 1-7 were identified in extracts of two bacterial Cytophaga strains (CFB-phylum) isolated from biofilms from the North Sea. Their structures are based on mono- and dimeric-cyclization products of 2-methylpropane-1,2-dithiol 8, which was also present in the extract in trace amounts. The structures were deduced by analysis of their mass spectra and confirmed by synthesis. The 1H NMR spectra of some these compounds suggested a high flexibility of the trithiepane and tetrathiocane systems. Therefore, their conformation was further analyzed by DFT calculations and dynamic NMR spectroscopy. While thiepane 4 possesses a twist-chair lowest energy conformation, its isomers 2 and 3 adopt a chairlike conformation, as does the tetrathiocane 5. In contrast, tetrathiocane 6 favors again a twisted chair conformation.

Cluster analysis as selection and dereplication tool for the identification of new natural compounds from large sample sets.

Cluster analysis of gas-chromatographic (GC) data of ca. 500 bacterial isolates was used as an aid in detection and identification of new natural compounds. This approach reduces the number of GC/MS analysis (dereplication) and concomitantly improves the selection of samples with high probability to contain unknown natural products. Lipophilic bacterial extracts were derivatized and analyzed by GC under standardized conditions. A program was developed to convert chromatographic data into a two-dimensional matrix. Based on the results of hierarchical cluster analysis samples were selected for further investigation by GC/MS and NMR. This approach avoided unnecessary analysis of similar samples. By this method, the unusual oligoprenylsesquiterpenes 1 and 2 as well as new aromatic amides 7 and 8 were identified.