Novel pheromone-mediated reproductive behaviour in the stag beetle, Lucanus cervus.

The iconic European stag beetle (Lucanus cervus) (Coleoptera: Lucanidae) is one of the largest terrestrial beetles in Europe. Due to decreasing population numbers, thought to be a consequence of habitat loss, this beetle has become a near-threatened species across much of Europe, and a reliable monitoring system is required to measure its future population trends. As part of a programme aimed at conserving UK populations, we have investigated the chemical ecology of the beetle, with a view to developing an efficient semiochemical-based monitoring system. Such a scheme will be beneficial not only in the UK but across the European range of the species, where the beetle is of conservation concern. Here, we report on a surprising discovery of a male-produced pheromone, which provokes initial sexual receptivity in females, and which has not been previously identified in the animal kingdom. Furthermore, we assign sex pheromone function to a previously described female-specific compound.

A diterpene synthase from the sandfly Lutzomyia longipalpis produces the pheromone sobralene.

The phlebotomine sandfly, Lutzomyia longipalpis, a major vector of the Leishmania parasite, uses terpene pheromones to attract conspecifics for mating. Examination of the L. longipalpis genome revealed a putative terpene synthase (TPS), which-upon heterologous expression in, and purification from, Escherichia coli-yielded a functional enzyme. The TPS, termed LlTPS, converted geranyl diphosphate (GPP) into a mixture of monoterpenes with low efficiency, of which ß-ocimene was the major product. (E,E)-farnesyl diphosphate (FPP) principally produced small amounts of (E)-ß-farnesene, while (Z,E)- and (Z,Z)-FPP yielded a mixture of bisabolene isomers. None of these mono- and sesquiterpenes are known volatiles of L. longipalpis. Notably, however, when provided with (E,E,E)-geranylgeranyl diphosphate (GGPP), LlTPS gave sobralene as its major product. This diterpene pheromone is released by certain chemotypes of L. longipalpis, in particular those found in the Ceará state of Brazil. Minor diterpene components were also seen as products of the enzyme that matched those seen in a sandfly pheromone extract.

Evaluating repellence properties of catnip essential oil against the mosquito species Aedes aegypti using a Y-tube olfactometer.

The mosquito species Aedes aegypti (L.) is known to act as a vector in the transmission of various diseases, including dengue fever and yellow fever. The use of insect repellents is one of precautionary measures used to mitigate the risk of these diseases in humans by reducing mosquito biting. Nepetalactone, a potent natural insect repellent primarily found in catnip (Nepeta cataria) essential oil, has emerged as a promising candidate for mosquito repellence. Here, we evaluated the potential of catnip essential oil (> 95% nepetalactone) for use as a mosquito repellent. Using a Y-tube olfactometer and human hands as an attractant, we analysed the effectiveness of catnip oil at repelling the mosquito species Aedes aegypti. We tested a range of dilutions of catnip essential oil and found that concentrations as low as 2% were effective at repelling > 70% of mosquitoes for between one and four hours after repellent application. These findings suggest that nepetalactone could potentially be used as a natural, effective alternative to synthetic mosquito repellents, thereby offering protection against vector-borne diseases.

Characterisation of geranylgeranyl diphosphate synthase from the sandfly Lutzomyia longipalpis.

Leishmaniasis is a debilitating and often fatal neglected tropical disease. Males from sub-populations of the Leishmania-harbouring sandfly, Lutzomyia longipalpis, produce the diterpene sex and aggregation pheromone, sobralene, for which geranylgeranyl diphosphate (GGPP) is the likely isoprenoid precursor. We have identified a GGPP synthase (lzGGPPS) from L. longipalpis, which was recombinantly expressed in bacteria and purified for functional and kinetic analysis. In vitro enzymatic assays using LC-MS showed that lzGGPPS is an active enzyme, capable of converting substrates dimethylallyl diphosphate (DMAPP), (E)-geranyl diphosphate (GPP), (E,E)-farnesyl diphosphate (FPP) with co-substrate isopentenyl diphosphate (IPP) into (E,E,E)-GGPP, while (Z,E)-FPP was also accepted with low efficacy. Comparison of metal cofactors for lzGGPPS highlighted Mg2+ as most efficient, giving increased GGPP output when compared against other divalent metal ions tested. In line with previously characterised GGPPS enzymes, GGPP acted as an inhibitor of lzGGPPS activity. The molecular weight in solution of lzGGPPS was determined to be ∼221 kDa by analytical SEC, suggesting a hexameric assembly, as seen in the human enzyme, and representing the first assessment of GGPPS quaternary structure in insects.

Small volatile lipophilic molecules induced belowground by aphid attack elicit a defensive response in neighbouring un-infested plants.

In pioneering studies on plant-aphid interactions, we have observed that Vicia faba plants infested by aphids can transmit signals via the rhizosphere that induce aboveground defence in intact, neighbouring plants. The aphid parasitoid Aphidius ervi is significantly attracted towards intact broad bean plants grown in a hydroponic solution previously harbouring Acyrtosiphon pisum-infested plants. To identify the rhizosphere signal(s) possibly mediating this belowground plant-plant communication, root exudates were collected using Solid-Phase Extraction (SPE) from 10-day old A. pisum-infested and un-infested Vicia faba plants hydroponically grown. To verify the ability of these root exudates to trigger defence mechanisms against the aphids we added them to V. fabae plants grown in hydroponic solution, and tested these plants in the wind-tunnel bioassay to assess their attractiveness towards the aphids' parasitoids A. ervi. We identified three small volatile lipophilic molecules as plant defence elicitors: 1-octen-3-ol, sulcatone and sulcatol, in SPE extracts of A. pisum-infested broad bean plants. In wind tunnel assays, we recorded a significant increase in the attractiveness towards A. ervi of V. faba plants grown in hydroponic solution treated with these compounds, compared to plants grown in hydroponic treated with ethanol (control). Both 1-octen-3-ol and sulcatol have asymmetrically substituted carbon atoms at positions 3 and 2, respectively. Hence, we tested both their enantiomers alone or in mixture. We highlighted a synergistic effect on the level of attractiveness towards the parasitoid when testing the three compounds together in respect to the response recorded against them singly tested. These behavioural responses were supported by the characterization of headspace volatiles released by tested plants. These results shed new light on the mechanisms underlying plant-plant communication belowground and prompt the use of bio-derived semiochemicals for a sustainable protection of agricultural crops.

A Horizon Scan to Support Chemical Pollution-Related Policymaking for Sustainable and Climate-Resilient Economies.

While chemicals are vital to modern society through materials, agriculture, textiles, new technology, medicines, and consumer goods, their use is not without risks. Unfortunately, our resources seem inadequate to address the breadth of chemical challenges to the environment and human health. Therefore, it is important we use our intelligence and knowledge wisely to prepare for what lies ahead. The present study used a Delphi-style approach to horizon-scan future chemical threats that need to be considered in the setting of chemicals and environmental policy, which involved a multidisciplinary, multisectoral, and multinational panel of 25 scientists and practitioners (mainly from the United Kingdom, Europe, and other industrialized nations) in a three-stage process. Fifteen issues were shortlisted (from a nominated list of 48), considered by the panel to hold global relevance. The issues span from the need for new chemical manufacturing (including transitioning to non-fossil-fuel feedstocks); challenges from novel materials, food imports, landfills, and tire wear; and opportunities from artificial intelligence, greater data transparency, and the weight-of-evidence approach. The 15 issues can be divided into three classes: new perspectives on historic but insufficiently appreciated chemicals/issues, new or relatively new products and their associated industries, and thinking through approaches we can use to meet these challenges. Chemicals are one threat among many that influence the environment and human health, and interlinkages with wider issues such as climate change and how we mitigate these were clear in this exercise. The horizon scan highlights the value of thinking broadly and consulting widely, considering systems approaches to ensure that interventions appreciate synergies and avoid harmful trade-offs in other areas. We recommend further collaboration between researchers, industry, regulators, and policymakers to perform horizon scanning to inform policymaking, to develop our ability to meet these challenges, and especially to extend the approach to consider also concerns from countries with developing economies. Environ Toxicol Chem 2023;42:1212-1228. © 2023 Crown copyright and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article is published with the permission of the Controller of HMSO and the King's Printer for Scotland.

L-DOPA functions as a plant pheromone for belowground anti-herbivory communication.

While mechanisms of plant-plant communication for alerting neighbouring plants of an imminent insect herbivore attack have been described aboveground via the production of volatile organic compounds (VOCs), we are yet to decipher the specific components of plant-plant signalling belowground. Using bioassay-guided fractionation, we isolated and identified the non-protein amino acid l-DOPA, released from roots of Acyrtosiphon pisum aphid-infested Vicia faba plants, as an active compound in triggering the production of VOCs released aboveground in uninfested plants. In behavioural assays, we show that after contact with l-DOPA, healthy plants become highly attractive to the aphid parasitoid (Aphidius ervi), as if they were infested by aphids. We conclude that l-DOPA, originally described as a brain neurotransmitter precursor, can also enhance immunity in plants.

(-)-Adaline from the Adalia Genus of Ladybirds Is a Potent Antagonist of Insect and Specific Mammalian Nicotinic Acetylcholine Receptors.

Ladybird beetles (Coleoptera: Coccinellidae) possess strong chemical defences that are secreted in response to stress and are also found on the coating of eggs, which are rich in alkaloids that are responsible for their toxicity to other species. Recent studies have shown that alkaloids from several species of ladybird beetle can target nicotinic acetylcholine receptors (nAChRs) acting as receptor antagonists. Here, we have explored the actions of (-)-adaline, found in the 2-spot (Adalia bipunctata) and 10-spot (Adalia decempunctata) ladybirds, on both mammalian (α1ß1γδ, α7, α4ß2, α3ß4) and insect nAChRs using patch-clamp of TE671 cells and locust brain neurons natively expressing nAChRs, as well as two-electrode voltage clamp of Xenopus laevis oocytes recombinantly expressing nAChRs. All nAChR subtypes were antagonised by (-)-adaline in a time-dependent, voltage-dependent and non-competitive manner with the lowest IC50s at rat α3ß4 (0.10 µM) and locust neuron (1.28 µM) nAChRs, at a holding potential of -75 mV. The data imply that (-)-adaline acts as an open channel blocker of nAChRs.

Genetic Manipulation of Biosynthetic Pathways in Mint.

In recent years, the study of aromatic plants has seen an increase, with great interest from industrial, academic, and pharmaceutical industries. Among plants attracting increased attention are the Mentha spp. (mint), members of the Lamiaceae family. Mint essential oils comprise a diverse class of molecules known as terpenoids/isoprenoids, organic chemicals that are among the most diverse class of naturally plant derived compounds. The terpenoid profile of several Mentha spp. is dominated by menthol, a cyclic monoterpene with some remarkable biological properties that make it useful in the pharmaceutical, medical, cosmetic, and cleaning product industries. As the global market for Mentha essential oils increases, the desire to improve oil composition and yield follows. The monoterpenoid biosynthesis pathway is well characterised so metabolic engineering attempts have been made to facilitate this improvement. This review focuses on the Mentha spp. and attempts at altering the carbon flux through the biosynthetic pathways to increase the yield and enhance the composition of the essential oil. This includes manipulation of endogenous and heterologous biosynthetic enzymes through overexpression and RNAi suppression. Genes involved in the MEP pathway, the menthol and carvone biosynthetic pathways and transcription factors known to affect secondary metabolism will be discussed along with non-metabolic engineering approaches including environmental factors and the use of plant growth regulators.

Skin microbiome alters attractiveness to Anopheles mosquitoes.

BACKGROUND: Some people produce specific body odours that make them more attractive than others to mosquitoes, and consequently are at higher risk of contracting vector-borne diseases. The skin microbiome can break down carbohydrates, fatty acids and peptides on the skin into volatiles that mosquitoes can differentiate. RESULTS: Here, we examined how skin microbiome composition of women differs in relation to level of attractiveness to Anopheles coluzzii mosquitoes, to identify volatiles in body odour and metabolic pathways associated with individuals that tend to be poorly-attractive to mosquitoes. We used behavioural assays to measure attractiveness of participants to An. coluzzii mosquitoes, 16S rRNA amplicon sequencing of the bacteria sampled from the skin and gas chromatography of volatiles in body odour. We found differences in skin microbiome composition between the poorly- and highly-attractive groups, particularly eight Amplicon Sequence Variants (ASVs) belonging to the Proteobacteria, Actinobacteria and Firmicutes phyla. Staphylococcus 2 ASVs are four times as abundant in the highly-attractive compared to poorly-attractive group. Associations were found between these ASVs and volatiles known to be attractive to Anopheles mosquitoes. Propanoic pathways are enriched in the poorly-attractive participants compared to those found to be highly-attractive. CONCLUSIONS: Our findings suggest that variation in attractiveness of people to mosquitoes is related to the composition of the skin microbiota, knowledge that could improve odour-baited traps or other next generation vector control tools.

Using trained dogs and organic semi-conducting sensors to identify asymptomatic and mild SARS-CoV-2 infections: an observational study.

BACKGROUND: A rapid, accurate, non-invasive diagnostic screen is needed to identify people with SARS-CoV-2 infection. We investigated whether organic semi-conducting (OSC) sensors and trained dogs could distinguish between people infected with asymptomatic or mild symptoms, and uninfected individuals, and the impact of screening at ports-of-entry. METHODS: Odour samples were collected from adults, and SARS-CoV-2 infection status confirmed using RT-PCR. OSC sensors captured the volatile organic compound (VOC) profile of odour samples. Trained dogs were tested in a double-blind trial to determine their ability to detect differences in VOCs between infected and uninfected individuals, with sensitivity and specificity as the primary outcome. Mathematical modelling was used to investigate the impact of bio-detection dogs for screening. RESULTS: About, 3921 adults were enrolled in the study and odour samples collected from 1097 SARS-CoV-2 infected and 2031 uninfected individuals. OSC sensors were able to distinguish between SARS-CoV-2 infected individuals and uninfected, with sensitivity from 98% (95% CI 95-100) to 100% and specificity from 99% (95% CI 97-100) to 100%. Six dogs were able to distinguish between samples with sensitivity ranging from 82% (95% CI 76-87) to 94% (95% CI 89-98) and specificity ranging from 76% (95% CI 70-82) to 92% (95% CI 88-96). Mathematical modelling suggests that dog screening plus a confirmatory PCR test could detect up to 89% of SARS-CoV-2 infections, averting up to 2.2 times as much transmission compared to isolation of symptomatic individuals only. CONCLUSIONS: People infected with SARS-CoV-2, with asymptomatic or mild symptoms, have a distinct odour that can be identified by sensors and trained dogs with a high degree of accuracy. Odour-based diagnostics using sensors and/or dogs may prove a rapid and effective tool for screening large numbers of people.Trial Registration NCT04509713 (clinicaltrials.gov).

Bumblebee electric charge stimulates floral volatile emissions in Petunia integrifolia but not in Antirrhinum majus.

The timing of volatile organic compound (VOC) emission by flowering plants often coincides with pollinator foraging activity. Volatile emission is often considered to be paced by environmental variables, such as light intensity, and/or by circadian rhythmicity. The question arises as to what extent pollinators themselves provide information about their presence, in keeping with their long co-evolution with flowering plants. Bumblebees are electrically charged and provide electrical stimulation when visiting plants, as measured via the depolarisation of electric potential in the stem of flowers. Here we test the hypothesis that the electric charge of foraging bumblebees increases the floral volatile emissions of bee pollinated plants. We investigate the change in VOC emissions of two bee-pollinated plants (Petunia integrifolia and Antirrhinum majus) exposed to the electric charge typical of foraging bumblebees. P. integrifolia slightly increases its emissions of a behaviorally and physiologically active compound in response to visits by foraging bumblebees, presenting on average 121 pC of electric charge. We show that for P. integrifolia, strong electrical stimulation (600-700 pC) promotes increased volatile emissions, but this is not found when using weaker electrical charges more representative of flying pollinators (100 pC). Floral volatile emissions of A. majus were not affected by either strong (600-700 pC) or weak electric charges (100 pC). This study opens a new area of research whereby the electrical charge of flying insects may provide information to plants on the presence and phenology of their pollinators. As a form of electroreception, this sensory process would bear adaptive value, enabling plants to better ensure that their attractive chemical messages are released when a potential recipient is present.

Iridoid Sex Pheromone Biosynthesis in Aphids Mimics Iridoid-Producing Plants.

Biosynthesis of (1R,4aS,7S,7aR)-nepetalactol (1) and (4aS,7S,7aR)-nepetalactone (2) in plants involves iridoid synthase (ISY), an atypical reductive cyclase that catalyses the reduction of 8-oxogeranial into the reactive enol of (S)-8-oxocitronellal, and cyclization of this enol intermediate, either non-enzymatically or by a nepetalactol-related short chain dehydrogenase enzyme (NEPS) that yields the nepetalactols. In this study, we investigated the biosynthesis in vivo of 1 and 2 in the pea aphid, Acyrthosiphon pisum, using a library of isotopically-labelled monoterpenoids as molecular probes. Topical application of deuterium-labelled probes synthesized from geraniol and nerol resulted in production of 2 H4 -lactol 1 and 2 H4 -lactone 2. However, deuterium incorporation was not evident using labelled probes synthesized from (S)-citronellol. These results suggest that iridoid biosynthesis in animals, specifically aphids, may follow a broadly similar route to that characterised for plants.

Sex Pheromone of the Alfalfa Plant Bug, Adelphocoris lineolatus: Pheromone Composition and Antagonistic Effect of 1-Hexanol (Hemiptera: Miridae).

The sex pheromone composition of alfalfa plant bugs, Adelphocoris lineolatus (Goeze), from Central Europe was investigated to test the hypothesis that insect species across a wide geographical area can vary in pheromone composition. Potential interactions between the pheromone and a known attractant, (E)-cinnamaldehyde, were also assessed. Coupled gas chromatography-electroantennography (GC-EAG) using male antennae and volatile extracts collected from females, previously shown to attract males in field experiments, revealed the presence of three physiologically active compounds. These were identified by coupled GC/mass spectrometry (GC/MS) and peak enhancement as hexyl butyrate, (E)-2-hexenyl butyrate and (E)-4-oxo-2-hexenal. A ternary blend of these compounds in a 5.4:9.0:1.0 ratio attracted male A. lineolatus in field trials in Hungary. Omission of either (E)-2-hexenyl-butyrate or (E)-4-oxo-2-hexenal from the ternary blend or substitution of (E)-4-oxo-2-hexenal by (E)-2-hexenal resulted in loss of activity. These results indicate that this Central European population is similar in pheromone composition to that previously reported for an East Asian population. Interestingly, another EAG-active compound, 1-hexanol, was also present in female extract. When 1-hexanol was tested in combination with the ternary pheromone blend, male catches were reduced. This compound showed a dose-response effect with small doses showing a strong behavioral effect, suggesting that 1-hexanol may act as a sex pheromone antagonist in A. lineolatus. Furthermore, when (E)-cinnamaldehyde was field tested in combination with the sex pheromone, there was no increase in male catch, but the combination attracted both males and females. Prospects for practical application are discussed.

Chemical Identity and Functional Characterization of Semiochemicals That Promote the Interactions between Rice Plant and Rice Major Pest Nilaparvata lugens.

The interaction between food crops and insect pests is mediated by semiochemicals emitted from host plants. These semiochemicals are natural behavioral modifiers and act on the insect olfactory system to locate hosts and preys. In this study, eight rice neuroactive semiochemicals were identified from rice varieties by GC-EAG and GC-MS. Their ability to modify rice pest behaviors was further studied as individual chemicals and physiologically relevant blend. The total amount of each semiochemical and the expression of their biosynthesis genes were significantly higher in pest susceptible variety than in pest-resistant variety and upregulated by the infestation of the pest Nilaparvata lugens (BPH). The semiochemicals emitted by uninfested plants (UIRVs) were more attractive to BPHs. Interestingly, the attractiveness of UIRVs was significantly reduced by the addition of the blend that mimics the natural composition of these semiochemicals emitted by infested plants (IRVs). Our study suggests a mechanism for the spread of pest infestation from infested plants to uninfested plants nearby. UIRVs initially serve as attractive signals to rice insect pests. The pest infestation changes the rice semiochemical profile to be less attractive or even repellent, which pushes further colonization to uninfested plants nearby. The identified semiochemicals can be used for crop protection based on a push-pull strategy.

Effects of Nicotine and Tobacco-Related Products on the Feeding Behavior of the German Cockroach (Blattodea: Blattellidae).

Animals use olfaction to detect developmentally significant volatile organic compounds (VOCs) in their local environment. As part of a wider study aiming to demonstrate that the olfactory responses of animals to VOCs can be modified through the creation of a drug-addicted status and association with a selected VOC, we investigated nicotine and tobacco smoke particulate (TSP) extract as possible addictive compounds for male German cockroaches, Blattella germanica (Linnaeus). In feeding experiments using an artificial food stimulus, food treated with TSP extract was preferred over untreated food. Surprisingly, nicotine, which was expected to be the most important addictive tobacco component, did not induce noticeable effects on cockroach behavior. Both TSP extract and nicotine were shown to be phagostimulants. Olfactometry assays that measured odor-mediated insect behavior demonstrated that male B. germanica did not choose TSP-extract-treated food even when attempts were made specifically to train them via this modality. These results support a hypothesis that B. germanica needs to consume TSP-containing food to show a clear preference for this stimulus and that gustatory mechanisms are involved due to compounds present in the TSP extract.

Overexpression of the homoterpene synthase gene, OsCYP92C21, increases emissions of volatiles mediating tritrophic interactions in rice.

Plant defence homoterpenes can be used to attract pest natural enemies. However, the biosynthetic pathway of homoterpenes is still unknown in rice, and the practical application of such indirect defence systems suffers from inherent limitations due to their low emissions from plants. Here, we demonstrated that the protein OsCYP92C21 is responsible for homoterpene biosynthesis in rice. We also revealed that the ability of rice to produce homoterpenes is dependent on the subcellular precursor pools. By increasing the precursor pools through specifically subcellular targeting expression, genetic transformation and genetic introgression, we significantly enhanced homoterpene biosynthesis in rice. The final introgressed GM rice plants exhibited higher homoterpene emissions than the wild type rice and the highest homoterpene emission reported so far for such GM plants even without the induction of herbivore attack. As a result, these GM rice plants demonstrated strong attractiveness to the parasitic wasp Cotesia chilonis. This study discovered the homoterpene biosynthesis pathway in rice, and lays the foundation for the utilisation of plant indirect defence mechanism in the "push-pull" strategy of integrated pest management through increasing precursor pools in the subcellular compartments and overexpressing homoterpene synthase by genetic transformation.

Three Aphid-Transmitted Viruses Encourage Vector Migration From Infected Common Bean (Phaseolus vulgaris) Plants Through a Combination of Volatile and Surface Cues.

Bean common mosaic virus (BCMV), bean common mosaic necrosis virus (BCMNV), and cucumber mosaic virus (CMV) are important pathogens of common bean (Phaseolus vulgaris), a crop vital for food security in sub-Saharan Africa. These viruses are vectored by aphids non-persistently, with virions bound loosely to stylet receptors. These viruses also manipulate aphid-mediated transmission by altering host properties. Virus-induced effects on host-aphid interactions were investigated using choice test (migration) assays, olfactometry, and analysis of insect-perceivable volatile organic compounds (VOCs) using gas chromatography (GC)-coupled mass spectrometry, and GC-coupled electroantennography. When allowed to choose freely between infected and uninfected plants, aphids of the legume specialist species Aphis fabae, and of the generalist species Myzus persicae, were repelled by plants infected with BCMV, BCMNV, or CMV. However, in olfactometer experiments with A. fabae, only the VOCs emitted by BCMNV-infected plants repelled aphids. Although BCMV, BCMNV, and CMV each induced distinctive changes in emission of aphid-perceivable volatiles, all three suppressed emission of an attractant sesquiterpene, α-copaene, suggesting these three different viruses promote migration of virus-bearing aphids in a similar fashion.