A variety of models have been used in mating bioassays of insects to assess the contribution of chemical and visual signals to mate location and mate selection. Although the use of such ‘dummies’ has had varying degrees of success, some insect species refuse to accept simplistic models. In the present study, we developed a 3D-printed model to explore whether more realistic models will be more successful than simplistic models in mating assays of difficult to manipulate species such as the flea beetle Altica fragariae. We ran five experiments to test (1) whether males could discriminate between males and females solely based on differences in cuticular hydrocarbons (CHCs), (2) whether males use shape or (3) color to choose mates, and (4) whether males can discriminate between 3D-printed models and freshly killed beetles either with or (5) without legs and antennae. The results of these experiments confirmed that male A. fragariae preferred models coated with CHCs of females over that of male CHCs, providing strong support for the role of CHCs in mate choice in Altica. We also showed that males use both shape and color in mate selection, and that males are capable of discriminating between the models and real beetle specimens. Together, the results indicate that 3D-printed models can provide a feasible and cost-effective method for mating studies of insects.

Luteolin 7-O-glucoside (1), 10β-acetoxy-8α-butyryloxy-11α-hydroxy-2β-((2-methylbutanoyl)oxy)-1βH,6αH,7αH,11βH-guaian-3-en-12,6-olide (2) and thapsigargin (3) herbicidal activities’ were evaluated in comparison with their binary and tertiary mixtures, against Lolium perenne. These allelochemicals were isolated from Thapsia garganica leaves methanolic extract. Experiments were carried out by irrigation and spray in pot trials. Each compound was tested at the concentration that inhibits 50% of L. perenne root growth (IC50). Mixtures were prepared at the total concentration determined to inhibit 50% of weed root growth based upon the IC50 value for each compound (1000 µM, 154 µM and 300 µM for 1, 2 and 3, respectively). The greatest herbicidal effect was observed in tertiary mixtures, followed by binary ones, and single compounds showed the lowest phytotoxicity. Moreover, spray treatment was more effective at inhibiting growth of L. perenne, compared with irrigation. For sprayed binary mixtures, the 2 and 3 mixture showed the best inhibitions in shoot (75.79%) and root (91.02%) growth, and fresh weight (89.28%). These values significantly improved those of the most active single compound, 1 (48.01%, 58.62% and 57.14%, respectively, following spray). On the other hand, compound 3, whose structure is related to guaianolide sesquiterpene lactones, was a common constituent of the most active mixtures, suggesting that it plays a more relevant role in the improvement of the phytotoxicity of mixtures. Results obtained for the spray treatment of the tertiary mixture of 1 (333.33 µM), 2 (51.33 µM) and 3 (100 µM) were even more prominent, since weed growth was completely inhibited. After irrigation with a tertiary mixture, the greatest inhibitions in shoot and root growth and fresh weight did not exceed 88.16%, 94% and 90.47%, respectively. The results reported highlight a synergistic behavior of the test allelochemicals which could be applied in the development of bio-herbicides.

Coniferous trees of the genus Pinus (Pinaceae) are under continuous threats by numerous herbivorous insect species and pathogens attacking nearly all parts and tissues of the plants. To defend themselves, pine trees produce large amounts of oleoresin that is accumulated in a highly developed network of specialized resin ducts, which are distributed in the wood, bark, and needles. Such defense reactions in pines can be induced by the attack of herbivores. The banded pine weevil, Pissodes castaneus (De Geer, 1775) (Coleoptera, Curculionidae), is an important pest of Pinus in Brazil, where it has been an invasive species since 2001. The female lays its eggs under the tree bark of trees and the larvae feed in the phloem of the trunk and branches, interrupting the sap circulation and eventually causing its death. In the present study, we conducted detailed GC–MS analyses of volatiles emitted by twigs of Pinus taeda L. We analyzed how the attack by P. castaneus males and females affects the volatile pattern emitted by the twigs. When comparing volatiles produced by healthy plants and by female- and male-attacked P. taeda, qualitative and quantitative differences were detected, as the decreased production of limonene, germacrene D and (E)-caryophyllene and the increase of α-pinene. Laboratory bioassays showed that plants attacked by male and female P. castaneus were more attractive to the insects. Understanding about what compounds may attract or repel the insects may help in the development of more effective traps, as well as preventing stress to avoid infestation.

The use of chemical pesticides as a main pest control strategy has been highly criticised due to environmental pollution and negative effects on natural enemies of pests. In modern farming, it is essential to implement integrated pest management approaches that seek to control insect pests without causing environmental damage, e.g. the use of companion plants. Basil and Mexican marigold are often used as companion plants to attract greenhouse whiteflies, hence reducing damage to solanaceous crops, but the mechanism and role of volatile cues in crop protection strategies are unknown. This study found that both flowering basil and marigold were preferred to tomato by the greenhouse whitefly (Trialeurodes vaporariorum) in Y-tube olfactometer bioassays. PCA revealed that some volatiles were more correlated to one stage than to another. The dominant volatile constituents of Mexican marigold are limonene, dihydrotagetone, (Z)-β-ocimene, α-pinene, (Z)-3-hexenyl acetate, and those from basil are linalool, 1,8-cineole, eugenol and β-elemene. Among these dominant compounds, 1,8-cineole and (Z)-3-hexenyl acetate elicited strong attraction in greenhouse whitefly at 0.01%, whereas (Z)-β-ocimene and linalool elicited strong repellence at 0.1% and 1% dosages. This suggested that the basil flowering stage attraction is due to 1,8-cineole. These volatiles demonstrated potential as lures or bio-repellents and could be used in a “push–pull” semiochemical approach for greenhouse whitefly management.

Cover crops provide many agroecosystem services, including weed suppression, which is partially exerted through release of allelopathic benzoxazinoid (BX) compounds. This research (1) characterizes changes in concentrations of BX compounds in shoots, roots, and soil at three growth stages (GS) of cereal rye (Secale cereale L.), and (2) their degradation in soil over time following termination. Concentrations of shoot dominant BX compounds, DIBOA-glc and DIBOA were lowest at GS 83 (boot). The root dominant BX compound, HMBOA-glc, concentration was least at GS 54 (elongation). Rhizosphere soil BX concentrations were 1000 times smaller than in root tissues. Dominant compounds in soil were HMBOA-glc and HMBOA. Soil BX compound concentrations were similar near root crowns and between-rows. Soil BX concentrations following cereal rye termination declined exponentially over time in three of four treatments: incorporated shoots (S) and roots (R), no-till S + R (cereal rye rolled flat), and no-till R (shoots removed); no-till S had consistently low concentrations. In treatments showing changes, soil concentrations of HMBOA-glc and HMBOA increased above initial concentrations on the day following cereal rye termination. Concentrations of these two compounds decreased more rapidly than the other compounds. Placement of shoots on the surface of an area where cereal rye had not grown (no-till S) did not increase soil concentrations of BX compounds. The short duration and complex dynamics of BX compounds in soil prior to and following termination illustrate the limited window for enhancing weed suppression directly by cereal rye allelochemicals; valuable information for programs breeding for enhanced weed suppression.

Nickel hyperaccumulation in Blepharidium guatemalense Standl. (Rubiaceae) was found in the tropical forests of south-eastern Mexico. This study aimed to document the geographic extent of nickel hyperaccumulation in this species, to understand its process of hyperaccumulation and to explore nickel distribution within the tissues of this plant. To accomplish these objectives, a complete non-destructive elemental screening of herbarium specimens was performed with a hand-held X-ray fluorescence spectrometer. Besides, rhizosphere soils and plant tissues were collected in Mexico and analyzed for physical–chemical parameters. Finally, elemental distribution maps of nickel and other elements in plant tissues were obtained by X-ray fluorescence spectroscopy and microscopy. This study revealed that Blepharidium guatemalense is distributed throughout Chiapas, Tabasco and Campeche, reaching the maximum nickel concentration in leaves (4.3 wt%) followed by roots and seeds (2.0 wt%) and bark (1.8 wt%). Simultaneous hyperaccumulation of cobalt and nickel was found in 15% of the herbarium specimens. Blepharidium guatemalense has uncommon re-distribution mechanisms via phloem since this tissue is the highest nickel-enriched from all parts of the plant (from roots to leaves). A high total nickel (mean of 610 µg g−1) was found in rhizosphere soils even though no evidence of ophiolite emplacement in that area has been reported. Blepharidium guatemalense represents the first hypernickelophore (> 1 wt% Ni) to be reported as growing in soils that are neither ultramafic nor enriched by anthropogenic pollutants.

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Quinones are one of the major pigment groups that provide such bright colors to feather stars (Echinodermata, Crinoidea). These secondary metabolites also act as defensive molecules rendering crinoids unpalatable and repellent to other organisms. However, feather stars are usually associated with numerous symbiotic organisms, amongst which the ectocommensal snapping shrimp Synalpheus stimpsonii. We investigated the chemical stimulus allowing host selection in S. stimpsonii through the combination of behavioral tests, chemical extractions, and mass spectrometry analyses. The individuals of S. stimpsonii used in the experiments were sampled around the Great Reef of Toliara (Madagascar) where they are found in association with two crinoid species: Comanthus wahlbergii and Phanogenia distincta. The chemical attractiveness of the two crinoid hosts and a non-host species, Cenometra bella, was tested in an olfactometer. The three crinoids produced attractive kairomones allowing the snapping shrimp to recognize them. Mass spectrometry analyses on purified extracts of P. distincta revealed the presence of three different anthraquinones (rhodoptilometrin, comantherin, and a new crinoid anthraquinone). Compared to the existing literature, this anthraquinonic cocktail is specific to P. distincta. When these extracts were injected in the olfactometer, they triggered similar attracting behavior suggesting that crinoid anthraquinones are kairomones allowing host selection for S. stimpsonii. This hypothesis is also supported by the fact that shrimps were chemically attracted by pure commercial anthraquinones. In addition to their traditional defensive role (allomones), anthraquinones would, therefore, also function as kairomones, maintaining the symbiosis between S. stimpsonii and its crinoid hosts.

Two components of the Synanthedon bicingulata sex pheromone, (E,Z)-3,13-octadecadienyl acetate (E3,Z13-18:OAc) and (Z,Z)-3,13-octadecadienyl acetate (Z3,Z13-18:OAc), were synthesized to investigate the effect of pheromone blends, trap type and trap color on the capture of S. bicingulata males. The optimal sex pheromone ratio for E3,Z13-18:OAc and Z3,Z13-18:OAc was approximately 4.3:5.7 based on the purity of the two pheromone components in all test areas. A significant difference was observed in the number of S. bicingulata adult males caught in bucket and delta traps. The mean numbers of males caught in bucket and delta traps were 13.2 ± 2.2 and 7.6 ± 2.0, respectively. Trap color affected the number of adult males caught in bucket traps. More adult males were attracted to a yellow bucket trap than to green, white, blue, black and red traps. An analysis of the relationship between trap capture and trap surface-color values (L*a*b*) revealed a positive relationship between trap capture and b* value.

The South American locust, Schistocerca cancellata (Serville 1838), is considered to be the most serious agricultural pest in Argentina and neighboring countries. The species is famous for the strong and pleasant aromatic scent of mature males in the gregarious phase. We identified this fragrance as a mixture of two main aromatic compounds, benzyl alcohol, and 2-phenylethanol. The male-specific volatiles are emitted almost exclusively from the abdomen. The release of the male volatiles is linked to sexual maturity and is influenced by the intensity of sexual competition between males. Consequently, a function of the volatiles as a gregarious mature male-emitted pheromone in the context of reproduction is obvious. No female-specific volatiles were found. Some compounds of the semivolatile fraction of the cuticular hydrocarbon (CHC) profile were also detected in the headspace of both sexes. This fraction of the CHCs of S. cancellata consists almost entirely of unbranched C23 to C31 alkanes. The profiles vary slightly between the sexes and differ from the reference profile of the prominent and well-studied species S. gregaria. Thus, semivolatile components of the CHCs may also play an important role in the short-range mate recognition system of the species.

In this work, we discussed and counter-commented Paul J. Weldon's comments on our recent paper (Zhou et al. Proc Natl Acad Sci USA 117:32493, 2020a), where we reported that BCP/BCPO (beta-caryophyllene/caryophyllene oxide) in fresh horse manure is sufficient to drive manure rolling behavior (HMR) in giant panda and attenuate the cold sensitivity of mice by directly targeting and inhibiting transient receptor potential melastatin 8 (TRPM8), an archetypical cold-activated ion channel of mammals. The main question we arise in this response is: “which is the reasonable target of BCP/BCPO? Parasites or TRPM8?” Based on the knowledge of TRPM8-mediated cooling sensation, interaction between BCP/BCPO and TRPM8, BCP/BCPO concentration in horse manure samples, correlation between HMR frequency and habitat temperature, insecticidal activity of BCP/BCPO and thermal ecology of parasites, we prefer a simple idea that BCP/BCPO-induced TRPM8 antagonism bestows the wild giant pandas with cold tolerance at low-ambient temperatures. Compared with the speculation of insecticidal activity induced by HMR behavior, our study provided a comprehensive mechanism to confirm a physiological target of BCP/BCPO during the highly cold-correlated behavior.

In social insects, chemical communication is the main communication mode among colony members, which use the blends of cuticular hydrocarbons as recognition cues to discriminate between nestmates and non-nestmates and to prevent the exploitation of their nest resources by aliens. The aim of this study was to assess the variation of nestmate recognition cues in the ant Ectatomma ruidum, a species complex with a considerably conserved morphology and one of the few ant species where intraspecific thievery, a form of cleptoparasitism, has been reported. We analyzed the cuticular hydrocarbon profiles of ants collected from a number of geographically separated populations and examined DNA sequence data to assess their species identity. We focused on one species of the complex, E. ruidum sp. 3–4, whose species delineation remains controversial. We documented that several quantitative and qualitative traits of the cuticular hydrocarbon profiles varied significantly between populations, indicating that this species harbors more cuticular chemical phenotypic diversity than expected within a single species. In particular, there was a striking divergence among populations in the proportion of methylalkanes, alkenes, alkadienes and odd-chain components, which likely play a major role in nestmate/non-nestmate discrimination, a process which might have been crucial in these cleptobiotic ants. Further investigations are needed to test the hypothesis that biotic pressures, such as the need to discriminate conspecific intruders and limit thievery, could have played an important role in promoting the evolutionary divergence between populations in this ant species complex.

In insects, chemical communication is the most common form of communication, and cuticular hydrocarbons (CHCs) are employed in recognition processes. In social insects, CHCs also help define colony identity and thus contribute to social cohesion among nestmates. Individuals can deposit their chemical signatures on nest surfaces. This information serves as a reference for newly emerged individuals and allows them to obtain the odor specific to their colony. This study examined nest chemical profiles in an inbred invasive species: the yellow-legged hornet, Vespa velutina nigrithorax. We demonstrated that nest structures (i.e., envelopes, combs, and pillars) had specific hydrocarbon profiles, which were colony specific. There were similarities between the chemical profiles of the nests and the CHC signatures of hornets. The loss of genetic diversity previously documented in the yellow-legged hornet population in France does not appear to have constrained nest chemical diversity.

Juvenile hormone (JH) is a key insect hormone involved in the regulation of physiological, developmental and behavioural processes. In social insects, it has been shown that JH can play a key role in modulating reproductive division of labour, age-related division of labour and chemical signalling, and can display marked changes in function of the degree of sociality. Here, we checked the effects of JH on reproduction in single foundresses of two neotropical primitively eusocial wasp species, Mischocyttarus cerberus and Mischocyttarus cassununga, by examining how treatments with the JH-analogue methoprene and the anti-JH precocene affect egg-laying, ovarian activation and chemical profiles. Our hypothesis was that reproduction and the production of particular fertility-linked cuticular hydrocarbon cues might be under shared JH control already in primitively eusocial wasp species, and this could have been a key enabler to allow such cues to later evolve into full-fledged queen pheromone signals in advanced eusocial species. In line with this hypothesis, we show that our hormone treatments significantly affected both egg laying and the production of particular hydrocarbons present on the egg surface. We discuss the relevance of these findings in the context of the evolution of social insect queen pheromones in advanced eusocial species with a morphologically differentiated queen–worker caste.

The scent gland secretion of an undetermined species of Prionostemma from Costa Rica was analyzed by gas chromatography–mass spectrometry and shown to consist of medium-chain carboxylic acids (mainly octanoic acid) and a ß-hydroxy-carboxylic acid, eventually identified as myrmicacin (= (R)-3-hydroxydecanoic acid). While scent gland secretions in harvestmen have traditionally been considered to be products of de novo synthesis, we here provide evidence for the unusual case of sequestration-derived scent gland constituents: at least myrmicacin appears to be sequestered from leaf-cutter ants that constitute a part of the prey of the Prionostemma-species herein investigated. This is the first report on the scent gland chemistry of the sclerosomatid subfamily Gagrellinae as well as on a possible sequestration mechanism in harvestmen.

Insect olfactory systems can efficiently distinguish important host signals in a complex background of odor. Notably, Conopomorpha sinensis Bradley (Lepidoptera: Gracillariidae), a host-specific pest of Litchi chinensis and Euphoria longan, causes periodic outbreaks in southern China. However, little is known about the functions of host volatiles and olfactory mechanisms through which C. sinensis senses host taxa. Consequently, the present study analyzed the Electroantennogram (EAG) responses of C. sinensis antennae to host volatile compounds and their mixtures. The results showed that volatile components were more stimulatory to female than to male C. sinensis antennae. In addition, the highest EAG responses were observed following the stimulation of female antennae by the individual volatile component β-guaiene, followed by β-caryophyllene and β-elemene. However, odorant mixtures containing β-farnesene and α-pinene significantly altered EAG responses in female antennae. This was further confirmed by behavioral responses to host volatile compounds based on the flight orientation of females and males in a wind tunnel. These findings demonstrated that the behavior of C. sinensis can be affected by single compounds or a mixture of compounds. Moreover, real-time fluorescence quantitative PCR suggested that a combination of α-pinene with β-farnesene could alter the expression of olfactory genes. Therefore, screening for odors that can effectively alter the behavior of insects provides a theoretical basis for exploring host recognition and utilizing the olfactory networks of C. sinensis for biocontrol, at the molecular level.

Spiroacetals such as E-7-methyl-1,6-dioxaspiro[4,5]decane (trans-conophthorin; tC) and acetamides [predominantly N-(3-methylbutyl)acetamide; N3MBA], are two major groups of volatiles discovered in venoms of many Vespidae. In the course of testing the attractiveness of tC and N3MBA to Vespidae using Rescue® Wasp TrapStiks, a significant number of female milichiids, Desmometopa nearctica Sabrosky and D. sordida (Fallén) (Diptera: Milichiidae) were trapped as well. However, the attraction of vespid wasps was not significant at the dosages tested. We found a significant synergistic effect of tC and N3MBA in attracting Desmometopa flies. Both D. nearctica and D. sordida are kleptoparasitic species; and we conclude that females of these two milichiid flies use tC and N3MBA (and likely other volatiles) released from venom glands of the social vespids (yellowjackets, paper wasps and hornets) as kairomones to locate disturbed, injured, or freshly killed insects (vespids and/or their prey) as a protein-rich food source for egg development and production.

The transition from a perennial to an annual life cycle, as well as domestication, are expected to increase plant growth and reproduction at the same time that anti-herbivore defences are reduced. Here, we investigated the effects of the life-history transition (the perennial teosinte Zea diploperennis to the annual teosinte Z. mays ssp. mexicana) and domestication of Zea (annual teosinte to the modern maize Z. mays ssp. mays) on direct and indirect defences against the fall armyworm Spodoptera frugiperda. The direct defence of Zea was assessed by larval survival and nutritional indices based on food intake and utilisation. Indirect defence was measured in terms of the olfactory preference of the night-active predatory earwig Doru luteipes for nocturnal herbivore-induced plant volatiles (HIPVs) from the teosintes and maize. Larval growth and survival were reduced on teosintes relative to maize. Whilst larvae fed on perennial teosinte had lower food intake indices, those on annual teosinte showed lower food utilisation indices relative to maize. The earwig preferred HIPVs emitted by teosintes over those by maize, but it did not discriminate between odours of herbivore-damaged annual and perennial teosinte. The nocturnal HIPV blend from maize contained the lowest total amount of fatty acid derivatives, while it had higher total amounts of terpenes compared to teosintes. Our study shows that the teosintes are better defended than maize in terms of direct and indirect defences; however, the perennial teosinte have stronger direct defences against the fall armyworm than the annual teosinte.

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Although recognition using cuticular chemistry is important for host–parasite interactions within aculeate Hymenoptera, cuticular hydrocarbon (CHC) profiles of only a few host–parasite pairs were characterized and compared. One largely neglected family in this context is the Mutillidae (velvet ants), whose species are ectoparasitoids of bees and wasps. In our study, we characterized and compared the CHC profiles of five species of Mutillidae and seven host species. The CHC profile of velvet ants differed among species and included large proportions of n-alkanes and methyl-branched alkanes. Alkenes were much less abundant in the CHC profiles of three species of velvet ants compared with their hosts, while the other two species possess a much lower abundance of methyl-branched alkanes than their hosts. Both the number of peaks and compound diversity were generally higher in velvet ants compared with their hosts. Thus, CHC profiles of parasitoids did not show signs of mimicry when compared with their hosts. In dyadic encounters between one species of velvet ant and its host bee species, the parasitoid mainly avoided interacting, while aggression by the host was rare. Our results suggest that velvet ants did not evolve chemical mimicry, perhaps in accordance with their wide host spectrum which would limit chemical specialization. However, the reduction of alkenes in social bee-attacking species and the reduction of methyl-branched alkanes in social wasp-attacking species may favour host nest invasion, since these two CHC classes are known to be important in nestmate recognition for social bees and wasps, respectively. A larger, phylogeny-corrected comparison of Mutillidae and hosts may help clarifying the evolution of the CHC profile of these parasitoids.