Binding properties of chemosensory protein 4 in Riptortus pedestris to aggregation pheromones.

In insects, chemosensory proteins (CSPs) play an important role in the perception of the external environment and have been widely used for protein-binding characterization. Riptortus pedestris has received increased attention as a potential cause of soybean staygreen syndrome in recent years. In this study, we found that RpedCSP4 expression in the antennae of adult R. pedestris increased with age, with no significant difference in expression level observed between males and females, as determined through quantitative real-time polymerase chain reaction (qRT-PCR). Subsequently, we investigated the ability of RpedCSP4 to bind various ligands (five aggregated pheromone components and 13 soybean volatiles) using a prokaryotic expression system and fluorescence competitive binding assays. We found that RpedCSP4 binds to three aggregated pheromone components of R. pedestris, namely, ((E)-2-hexenyl (Z)-3-hexenoate (E2Z3), (E)-2-hexenyl (E)-2-hexenoate (E2E2), and (E)-2-hexenyl hexenoate (E2HH)), and that its binding capacities are most stable under acidic condition. Finally, the structure and protein-ligand interactions of RpedCSP4 were further analyzed via homology modeling, molecular docking, and targeted mutagenesis experiments. The L29A mutant exhibited a loss of binding ability to these three aggregated pheromone components. Our results show that the olfactory function of RpedCSP4 provides new insights into the binding mechanism of RpedCSPs to aggregation pheromones and contributes to discover new target candidates that will provide a theoretical basis for future population control of R. pedestris.

Can Pheromones Contribute to Phylogenetic Hypotheses? A Case Study of Chrysomelidae.

Pheromones mediate species-level communication in the search for mates, nesting, and feeding sites. Although the role of pheromones has long been discussed by various authors, their existence was not proven until the mid-twentieth century when the first sex pheromone was identified. From this finding, much has been speculated about whether this communication mechanism has acted as a regulatory agent in the process of speciation, competition, and sexual selection since it acts as an intraspecific barrier. Chrysomelidae is one of the major Phytophaga lineages, with approximately 40,000 species. Due to this immense diversity the internal relationships remain unstable when analyzed only with morphological data, consequently recent efforts have been directed to molecular analyses to establish clarity for the relationships and found their respective monophyly. Therefore, our goals are twofold 1) to synthesize the current literature on Chrysomelidae sex pheromones and 2) to test whether Chrysomelidae sex pheromones and their chemical structures could be used in phylogenetic analysis for the group. The results show that, although this is the first analysis in Chrysomelidae to use pheromones as a phylogenetic character, much can be observed in agreement with previous analyses, thus confirming that pheromones, when known in their entirety within lineages, can be used as characters in phylogenetic analyses, bringing elucidation to the relationships and evolution of organisms.

Aggregation Pheromones of Weevils (Coleoptera: Curculionidae): Advances in the Identification and Potential Uses in Semiochemical-Based Pest Management Strategies.

With approximately 83,000 species described, Curculionidae is the largest family of beetles, comprising more than 80% of all weevil species worldwide. Many species of Curculionidae attack a wide range of native and orchards crops, as well as globally important stored products such as grains, flour, and seeds, being responsible for significant environmental and economic losses. This work provides an overview of the research in the identification of aggregation pheromones of Curculionidae, and their potential contributions to the development of semiochemical-based pest management strategies. The synergistic effect of the host plant volatiles in the attractiveness of weevil pheromones is also briefly reported, demonstrating the important role of these additional attractants in the chemical communication of curculionids.

Management of Western North American Bark Beetles with Semiochemicals.

We summarize the status of semiochemical-based management of the major bark beetle species in western North America. The conifer forests of this region have a long history of profound impacts by phloem-feeding bark beetles, and species such as the mountain pine beetle (Dendroctonus ponderosae) and the spruce beetle (D. rufipennis) have recently undergone epic outbreaks linked to changing climate. At the same time, great strides are being made in the application of semiochemicals to the integrated pest management of bark beetles. In this review, we synthesize and interpret these recent advances in applied chemical ecology of bark beetles for scientists and land managers.

Identification of male-produced aggregation pheromone of the curculionid beetle Acrotomopus atropunctellus.

The sugarcane stem weevil, Acrotomopus atropunctellus (Boheman) (Curculionidae: Molytinae: Cholini) is an important economic pest from the Northwestern region of Argentina. Analyses of the headspace volatiles produced by separated males and females revealed one male-specific compound. Its structural identification is reported here in using gas chromatography coupled with mass spectroscopy analysis and chemical micro-reactions. Besides, two laboratory olfactometry assays allowed us to propose 6-methyl-5-hepten-2-one (sulcatone) as an aggregation pheromone for this insect, being attractive to both conspecific males and females. This compound is reported for the first time as involved in the Curculionidae family communication.

Identification of a self-regulatory pheromone system that controls nymph aggregation behavior of rice spittlebug Callitettix versicolor.

BACKGROUND: Nymphs of many spittlebug species are known to aggregate in one spittle mass, a behavior which greatly benefits the survival of the developing nymphs. Little is known, however, about the precise mechanisms that induce and regulate aggregation. Here, we investigated the aggregation behavior of nymphs of the rice spittlebug Callitettix versicolor, and analyzed the chemical composition of spittle masses. RESULTS: We identified six n-alkane compounds, namely un-, do-, tri-, tetra-, penta- and hexadecane in the spittle mass. Importantly, we showed that solitary spittle mass (SSM) and aggregation spittle mass (ASM) differed significantly in the amounts and composition of these compounds. While un-, do-, tri-, tetra-and hexadecane were overrepresented in SSM, pentadecane was found at significantly higher levels in ASM. Electrophysiological experiments showed that antennae responses to these six compounds were significantly higher than to both the hexane and the docosane control, which suggests a specific role of the six volatile alkanes as pheromones. In agreement with this hypothesis, behavioral tests revealed that five of the six compounds (e.g. un-, do-, tri-, tetra-, and hexadecane) acted as attractants across a wide concentration range. Thus, these five compounds allow recruitment of additional nymphs to a growing spittle mass. The sixth compound, pentadecane, attracted nymphs at low doses, whereas at higher doses, this effect vanished, suggesting that this alkane functioned as a repellent, thus preventing recruitment of additional individuals to a full aggregation in a spittle mass. CONCLUSIONS: In summary, our study identified a simple, yet fully functional feedback mechanism which allows aggregation at low nymph numbers, while preventing over-crowding beyond a set number of nymphs within one spittle mass. In conclusion, our study provides new insights into C. versicolor development and behavior that should greatly facilitate the identification of new approaches for pheromonal control of this pest.

Pollination of Specklinia by nectar-feeding Drosophila: the first reported case of a deceptive syndrome employing aggregation pheromones in Orchidaceae.

BACKGROUND AND AIMS: The first documented observation of pollination in Pleurothallidinae was that of Endrés, who noticed that the 'viscid sepals' of Specklinia endotrachys were visited by a 'small fly'. Chase would later identify the visiting flies as being members of the genus Drosophila. This study documents and describes how species of the S. endotrachys complex are pollinated by different Drosophila species. METHODS: Specimens of Specklinia and Drosophila were collected in the field in Costa Rica and preserved in the JBL and L herbaria. Flies were photographed, filmed and observed for several days during a 2-year period and were identified by a combination of non-invasive DNA barcoding and anatomical surveys. Tissue samples of the sepals, petals and labellum of Specklinia species were observed and documented by SEM, LM and TEM. Electroantennogram experiments were carried out on Drosophila hydei using the known aggregation pheromones ethyl tiglate, methyl tiglate and isopropyl tiglate. Floral compounds were analysed by gas chromatography-mass spectometry using those same pheromones as standards. KEY RESULTS: Flowers of S. endotrachys, S. pfavii, S. remotiflora and S. spectabilis are visited and pollinated by several different but closely related Drosophila species. The flies are arrested by aggregation pheromones, including ethyl tiglate, methyl tiglate and isopropyl tiglate, released by the flowers, and to which at least D. hydei is very sensitive. Visible nectar drops on the adaxial surface of sepals are secreted by nectar-secreting stomata, encouraging male and female Drosophila to linger on the flowers for several hours at a time. The flies frequently show courtship behaviour, occasionally copulating. Several different Drosophila species can be found on a single Specklinia species. CONCLUSIONS: Species of the S. endotrachys group share a similar pollination syndrome. There seem to be no species-specific relationships between the orchids and the flies. It is not expected that Specklinia species will hybridize naturally as their populations do not overlap geographically. The combination of pheromone attraction and nectar feeding is likely to be a generalized pollination syndrome in Pleurothallidinae.

Ophiostomatoid fungi synergize attraction of the Eurasian spruce bark beetle, Ips typographus to its aggregation pheromone in field traps.

Eurasian spruce bark beetle, Ips typographus is a destructive pest of the Norway spruce (Picea abies). Recent outbreaks in Europe have been attributed to global warming and other anthropogenic impacts. Bark beetles are guided by multiple complex olfactory cues throughout their life cycle. Male-produced aggregation pheromones, comprising 2-methyl-3-buten-2-ol and cis-verbenol, have been identified as the most powerful attractants for dispersing conspecifics. In addition to host trees, bark beetles interact with multiple organisms, including symbiotic ophiostomatoid fungi, which may promote beetle colonization success and offspring development. Previously, in a short-distance laboratory assay, we demonstrated that I. typographus adults are attracted to the volatile organic compounds (VOCs) produced by three symbiotic fungi: Grosmannia penicillata, Endoconidiophora polonica, and Leptographium europhioides. Furthermore, the abundant fusel alcohols and their acetates were found to be the most attractive odorants in the fungal VOC profile. In this study, using a long-distance field-trapping experiment, we analyzed the role of fungal VOCs as attractants for dispersing I. typographus. Two types of fungal lures were tested in combination with pheromones in traps: (1) live cultures of fungi grown on potato dextrose agar (PDA) and (2) dispensers containing synthetic fusel alcohols and their acetates in equal proportions. Subsequently, the composition of VOCs emitted from live fungal lures were analyzed. We found that the symbiotic fungi synergistically increased the attraction of beetles to pheromones in field traps and the attractiveness of live fungal lures depended on the fungal load. While one Petri dish with E. polonica, when combined with pheromones synergistically increased trapping efficiency, three Petri dishes with L. europhioides were required to achieve the same. The synthetic mix of fungal fusel alcohols and acetates improved the catch efficiency of pheromones only at a low tested dose. VOC analysis of fungal cultures revealed that all the three fungi produced fusel alcohols and acetates but in variable composition and amounts. Collectively, the results of this study show that, in addition to pheromones, bark beetles might also use volatile cues from their symbiotic fungi to improve tree colonization and reproductive success in their breeding and feeding sites.

Attraction of Male Nymphs to Adult Male Volatiles in the Bronze Bug Thaumastocoris peregrinus Carpintero & Dellape (Heteroptera: Thaumastocoridae).

The bronze bug, Thaumastocoris peregrinus Carpintero & Dellape (Heteroptera: Thaumastocoridae), is an exotic emerging pest in Eucalyptus commercial forests in South America, Africa and southern Europe. Information on the chemical communication system and reproductive ecology of this insect is scant, and it may be relevant for designing management strategies for eucalypt plantations. Adults and nymphs usually aggregate in the field, possibly by means of chemical signals. Males emit large amounts of 3-methyl-2-butenyl butyrate, which attracts conspecific adult males but not females. The ecological role of this putative male aggregation pheromone remains unknown. Here, we report olfactometer bioassays showing that late-instar male nymphs are also attracted to synthetic 3-methyl-2-butenyl butyrate and to adult male volatile extracts, which contain this compound as the major component. As previously shown for adult females, nymphs that moulted into females were not attracted to either volatile stimulus. The intra-gender attraction of nymphs and adults may be related to the exploitation of food resources, or as a reproductive strategy for newly emerged males. Further studies on the reproductive behaviour and mating system of T. peregrinus will contribute to understanding the ecological significance of male-male, adult-nymph attraction, as well as the practical applications that may result from these findings.

Area-wide mass trapping by pheromone-based attractants for the control of sugar beet weevil (Bothynoderes punctiventris Germar, Coleoptera: Curculionidae).

BACKGROUND: Owing to the changing climate, narrow crop rotation, and changes in insecticide application practice, sugar beet weevil (SBW) (Bothynoderes punctiventris Germar) has become the most important economic pest in sugar beet. To develop alternative control methods, an area-wide (AW) control program using aggregation pheromones was implemented over 4 years on an area of 6 and 14.8 km2 in east Croatia. RESULTS: The mass trapping of SBW on the 'old' sugar beet fields reduced the population from 0.73% to 11.59%. Owing to the strong attack, mass trapping was not effective enough to avoid an insecticide application. However, it significantly reduced the number of insecticide applications, the amount of insecticide used, and the damage compared to the fields outside the mass trapping area. CONCLUSION: This is the first study to implement an AW program for SBW. It may not be possible to state from this study that trapping alone can reduce the SBW population below the economic threshold level. However, the data do suggest that trapping can play an important role in the reduction of insecticide applications and in creating an integrated pest management plan for dealing with SBW under similar circumstances. © 2017 Society of Chemical Industry.

Sexual chemoecology of mosquitoes (Diptera, Culicidae): Current knowledge and implications for vector control programs.

Mosquitoes (Diptera: Culicidae) act as vectors of medical and veterinary importance, due to their ability to transmit many pathogens and parasites. Renewed interest has been recently devoted to the potential of sterile insect technique (SIT) for mosquito suppression. However, the success of the SIT is mostly dependent on the ability of sterile males to compete for mates with the wild ones in the field. Nevertheless, little is known on the sexual chemical ecology of mosquitoes, with special reference to the role of chemical signals in males. We reviewed the current knowledge on mosquito sexual chemical ecology and other key cues affecting courtship and mating behavior. The information available on the aggregation and sex pheromones in mosquito males is rather limited. To the best of our knowledge, the components of the aggregation pheromone stimulating swarming mechanisms have been fully characterized only for Aedes aegypti, while evidence for aggregation pheromones in other mosquito species remains elusive. Further research on this issue is needed, as well as to dissect the relative importance of visual (with special reference to swarming landmarks), vibrational, olfactory and tactile cues perceived during swarming and mate. On the other hand, more knowledge is available for cuticular hydrocarbons, which modulate mating behavior in several species of economic importance. These compounds, coupled with volatile aggregation components, have potential interest for the development of monitoring and trapping systems. In addition, the analyses of cuticular hydrocarbons are essential for discrimination between closely related mosquito species and/or populations.

Swarming mechanisms in the yellow fever mosquito: aggregation pheromones are involved in the mating behavior of Aedes aegypti.

Mosquitoes of various species mate in swarms comprised of tens of thousands of flying males. In this study, we examined Aedes aegypti swarming behavior and identified associated chemical cues. Novel evidence is provided that Ae. aegypti females aggregate by means of olfactory cues, such as aggregation pheromones. Isolation of Ae. aegypti aggregation pheromones was achieved by aeration of confined mosquitoes and collection of associated volatiles by glass filters. The collected volatiles were identified through gas chromatography mass spectrometry (GCMS). Three aggregation pheromones were collected and identified as 2,6,6-trimethylcyclohex-2-ene-1,4-dione (ketoisophorone) (CAS# 1125-21-9, t(R) = 18.75), 2,2,6-trimethylcyclohexane-1,4-dione (the saturated analog of ketoisophorone) (CAS# 20547-99-3, t(R) = 20.05), and 1-(4-ethylphenyl) ethanone (CAS# 937-30-4, t(R) = 24.22). Our biological studies revealed that the identified compounds stimulated mosquito behavior under laboratory conditions. The mechanism of mosquito swarm formation is discussed in light of our behavioral study findings. A preliminary field trial demonstrated the potential application of the isolated aggregation pheromones in controlling Ae. aegypti.

Pheromonal Communication in the European House Dust Mite, Dermatophagoides pteronyssinus.

Despite the sanitary importance of the European house dust mite Dermatophagoides pteronyssinus (Trouessart, 1897), the pheromonal communication in this species has not been sufficiently studied. Headspace analysis using solid phase micro extraction (SPME) revealed that nerol, neryl formate, pentadecane, (6Z,9Z)-6,9-heptadecadiene, and (Z)-8-heptadecene are released by both sexes whereas neryl propionate was released by males only. Tritonymphs did not produce any detectable volatiles. In olfactometer experiments, pentadecane and neryl propionate were attractive to both sexes as well as to tritonymphs. (Z)-8-heptadecene was only attractive to male mites. Therefore it is discussed that pentadecane and neryl propionate are aggregation pheromones and (Z)-8-heptadecene is a sexual pheromone of the European house dust mite D. pteronyssinus. To study the potential use of pheromones in dust mite control, long-range olfactometer experiments were conducted showing that mites can be attracted to neryl propionate over distances of at least 50 cm. This indicates that mite pheromones might be useable to monitor the presence or absence of mites in the context of control strategies.