Developments in Fatty Acid-Derived Insect Pheromone Production Using Engineered Yeasts

The use of traditional chemical insecticides for pest control often leads to environmental pollution and a decrease in biodiversity. Recently, insect sex pheromones were applied for sustainable biocontrol of pests in fields, due to their limited adverse impacts on biodiversity and food safety compared to that of other conventional insecticides. However, the structures of insect pheromones are complex, and their chemical synthesis is not commercially feasible. As yeasts have been widely used for fatty acid-derived pheromone production in the past few years, using engineered yeasts may be promising and sustainable for the low-cost production of fatty acid-derived pheromones. The primary fatty acids produced by Saccharomyces cerevisiae and other yeasts are C16 and C18, and it is also possible to rewire/reprogram the metabolic flux for other fatty acids or fatty acid derivatives. This review summarizes the fatty acid biosynthetic pathway in S. cerevisiae and recent progress in yeast engineering in terms of metabolic engineering and synthetic biology strategies to produce insect pheromones. In the future, insect pheromones produced by yeasts might provide an eco-friendly pest control method in agricultural fields.

Production of Volatile Moth Sex Pheromones in Transgenic Nicotiana benthamiana Plants

Plant-based bioproduction of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here, we describe the engineering of transgenic plants producing (Z)-11-hexadecenol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them into Nicotiana benthamiana plants. The constructs contained the Amyelois transitella AtrΔ11 desaturase gene, the Helicoverpa armigera fatty acyl reductase HarFAR gene, and the Euonymus alatus diacylglycerol acetyltransferase EaDAct gene in different configurations. All the pheromone-producing plants showed dwarf phenotypes, the severity of which correlated with pheromone levels. All but one of the recovered lines produced high levels of Z11-16OH, but very low levels of Z11-16OAc, probably as a result of recurrent truncations at the level of the EaDAct gene. Only one plant line (SxPv1.2) was recovered that harboured an intact pheromone pathway and which produced moderate levels of Z11-16OAc (11.8 μg g-1 FW) and high levels of Z11-16OH (111.4 μg g-1). Z11-16OAc production was accompanied in SxPv1.2 by a partial recovery of the dwarf phenotype. SxPv1.2 was used to estimate the rates of volatile pheromone release, which resulted in 8.48 ng g-1 FW per day for Z11-16OH and 9.44 ng g-1 FW per day for Z11-16OAc. Our results suggest that pheromone release acts as a limiting factor in pheromone biodispenser strategies and establish a roadmap for biotechnological improvements.

Development of methods for application of pheromonic materials for monitoring and managing the number of apple phytophages

Synthetic sex pheromones have found wide application in plant protection as a mean of early detection of pests that allows observation of the phenology of insects to optimise protective measures. Insect sex pheromones can be applied for different purposes in protecting crops such as monitoring, determining the species composition and combating harmful species (by using sex pheromones for disorientation). The present work studies the species composition, dynamics of the number of basic pests of an apple tree, synchronisation of the seasonal and circadian activity of phytophages of an apple tree in the central zone of the Krasnodar Territory. The results of the field assessment of the male complex disorientation method of apple and eastern moths are presented. It was shown that the species-specificity of sex pheromones in the apple orchard depends on the faunistic diversity of Lepidoptera species with similar pheromone systems that develop at a given point in space and time. It was revealed that the behaviour of this complex changes during the season and over years, depending on the climate and natural dynamics of insect populations. The quantitative ratio and species-specificity of pheromones will probably be different in ecosystems with various species composition and different geographic zones. A novelty of this research is the division of the studied Lepidoptera phytophage species into three groups according to a decrease in the absolute species-specificity in the forest biotope in comparison with the garden one. The most widespread and coinciding in terms of summer synchronicity are apple (Cydia pomonella L.), plum (Grapholitha funebrana Tr.), eastern (Grapholitha molesta Tr.) and pomegranate moth (Euzophera bigella Zell.). The disorientation method, applied using a complex system of dispensers with apple and eastern moth pheromones, showed that installing 500 dispensers/ha allowed 99.3 % efficiency to be achieved. Fruit damage amounted to 1.2 % and 2.7 % in the experimental and control groups, respectively. The duration of the disorienting effect of the pheromone formulations lasted for over 4 months.

Host-Plant Volatiles Enhance the Attraction of Cnaphalocrocis medicals (Lepidoptera: Crambidae) to Sex Pheromone

Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae) is a notorious pest of rice, Oryza sativa L.(Poaceae). Sex pheromone and host-plant volatiles can trap C. medinalis separately. To improve the trap efficiency of sex pheromone, we first tested the synergistic effect of 8 host-plant volatiles, including 2-Phenylethanol, 1-Hexanol, 1-Heptanol, (Z)-3-Hexenal, (E)-2-Hexenal, Octanal, Valeraldehyde, and Methyl Salicylate on the attraction of C. medinalis to the female-produced sex pheromone in electroantennography. The addition of (E)-2-Hexenal, Methyl Salicylate, Valeraldehyde, and (Z)-3-Hexenal increased electroantennogram response of C. medinalis to sex pheromone. Further testing of the mixtures of these four compounds and sex pheromone in wind tunnel experiments indicated that additive (E)-2-Hexenal or Methyl Salicylate stimulated the landing behaviors of both male and female C. medinalis compared with sex pheromone alone. Field evaluations showed that mixtures of sex pheromone and (E)-2-Hexenal or Methyl Salicylate resulted in significantly higher catches to male moths than sex pheromone alone. Using 1:1 and 1:10 combinations of the sex pheromone and (E)-2-Hexenal, showed a synergistic effect of 95% and 110%, respectively. Furthermore, 1:1 and 1:10 mixtures of the sex pheromone and Methyl Salicylate exhibited a synergistic effect of 69% and 146%, respectively. These results may provide the basis for developing efficient pest management strategies against C. medinalis using host-plant volatiles and insect sex pheromones.

Production of Volatile Moth Sex Pheromones in Transgenic Nicotiana benthamiana Plants

Plant-based bio-production of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here we describe the engineering of transgenic plants producing (Z)-11-hexadecen-1-ol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multiple multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them in Nicotiana benthamiana plants. The constructs comprised the Amyelois transitella AtrΔ11 desaturase gene, the Helicoverpa armigera farnesyl reductase HarFAR gene, and the Euonymus alatus diacylglycerol acetyltransferase EaDAct gene in different configurations. All the pheromone-producing plants showed dwarf phenotypes, whose severity correlated with pheromone levels. All but one of the recovered lines produced high levels of Z11-16OH but very low levels of Z11-16OAc, probably as a result of recurrent truncations at the level of the EaDAct gene. Only one plant line (SxPv1.2) was recovered harbouring an intact pheromone pathway and producing moderate levels of Z11-16OAc (11.8 μg g-1 FW), next to high levels of Z11-16OH (111.4 μg g-1). Z11-16OAc production was accompanied in SxPv1.2 by a partial recovery of the dwarf phenotype. SxPv1.2 was used to estimate the rates of volatile pheromone release, which resulted in 8.48 ng g-1 FW per day for Z11-16OH and 9.44 ng g-1 FW per day for Z11-16OAc. Our results suggest that pheromone release acts as a limiting factor in pheromone bio-dispenser strategies and establish a roadmap for biotechnological improvements.

Production of moth sex pheromones for pest control by yeast fermentation

The use of insect sex pheromones is an alternative technology for pest control in agriculture and forestry, which, in contrast to insecticides, does not have adverse effects on human health or environment and is efficient also against insecticide-resistant insect populations.1,2 Due to the high cost of chemically synthesized pheromones, mating disruption applications are currently primarily targeting higher value crops, such as fruits.3 Here we demonstrate a biotechnological method for the production of pheromones of economically important moth pests using engineered yeast cell factories. Biosynthetic pathways towards several pheromones or their precursors were reconstructed in the oleaginous yeast Yarrowia lipolytica, which was further metabolically engineered for improved pheromone biosynthesis by decreasing fatty alcohol degradation and downregulating storage lipid accumulation. The sex pheromone of the cotton bollworm Helicoverpa armigera was produced by oxidation of fermented fatty alcohols into corresponding aldehydes. The resulting pheromone was just as efficient and specific for trapping of H. armigera male moths in cotton fields in Greece as a synthetic pheromone mixture. We further demonstrated the production of the main pheromone component of the fall armyworm Spodoptera frugiperda. Our work describes a biotech platform for the production of commercially relevant titres of moth pheromones for pest control by yeast fermentation.Significance statementAgriculture largely relies on insecticides and genetically modified crops for pest control, however alternative solutions are required due to emerging resistance, toxicity and regulatory issues, and consumer preferences. Mating disruption with sex pheromones that act by preventing insect reproduction is considered the most promising and scalable alternative to insecticides. This method is highly efficient and safe for human health and environment. The likelihood of insect resistance development is very low and can be handled by adjusting the pheromone composition. The high cost of chemically synthesized pheromones is the major barrier for the wider adoption of pheromones. A novel method based on yeast fermentation enables the production of insect sex pheromones as a lower cost from renewable feedstocks.

The Impact of Environmental Factors on the Efficacy of Chemical Communication in the Burying Beetle (Coleoptera: Silphidae)

There is growing evidence that a wide range of insect sex pheromones are condition dependent and play a fundamental role in mate choice. However, the effectiveness of pheromonal communication might not only depend on internal factors of the sender, but also on attributes of the microhabitat, in which the signaler chooses to emit its chemical signal. For example, the degree of anthropogenic land use might affect how successful the signal is transmitted, as land use has been shown to affect animal communities and the complexity of biotic interactions. To test the hypothesis that parameters of the microenvironment determine males’ ability to attract females via their sex pheromone, we used the burying beetle Nicrophorus vespilloides Herbst (Coleoptera: Silphidae) as our model system. We exposed 144 males across differently managed forest stands and analyzed the impact of 29 environmental parameters. Our data revealed that human land use intensity had no effect on a male’s attractiveness. However, the harvested tree biomass positively affected the proportion of competitors attracted. Furthermore, we found that soil characteristics were important factors determining the amount and body size of females a male was able to attract. Consequently, we present evidence that the environmental context of a signaling male influences the effectiveness of chemical signaling either because it affects the transmission process or the prevailing abundance of potential signal receivers. Thus, our results demonstrate that males need to make careful decisions about the location where they emit their pheromone, as this choice of microhabitat has an impact on their fitness.

Defense of Scots pine against sawfly eggs (Diprion pini) is primed by exposure to sawfly sex pheromones

Plants respond to insect infestation with defenses targeting insect eggs on their leaves and the feeding insects. Upon perceiving cues indicating imminent herbivory, such as damage-induced leaf odors emitted by neighboring plants, they are able to prime their defenses against feeding insects. Yet it remains unknown whether plants can amplify their defenses against insect eggs by responding to cues indicating imminent egg deposition. Here, we tested the hypothesis that a plant strengthens its defenses against insect eggs by responding to insect sex pheromones. Our study shows that preexposure of Pinus sylvestris to pine sawfly sex pheromones reduces the survival rate of subsequently laid sawfly eggs. Exposure to pheromones does not significantly affect the pine needle water content, but results in increased needle hydrogen peroxide concentrations and increased expression of defense-related pine genes such as SOD (superoxide dismutase), LOX (lipoxygenase), PAL (phenylalanine ammonia lyase), and PR-1 (pathogenesis related protein 1) after egg deposition. These results support our hypothesis that plant responses to sex pheromones emitted by an herbivorous insect can boost plant defensive responses to insect egg deposition, thus highlighting the ability of a plant to mobilize its defenses very early against an initial phase of insect attack, the egg deposition.