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.

Diel Patterns of Pheromone Release By Male Sea Lamprey

Costs to producing sexual signals can create selective pressures on males to invest signaling effort in particular contexts. When the benefits of signaling vary consistently across time, males can optimize signal investment to specific temporal contexts using biological rhythms. Sea lamprey, Petromyzon marinus, have a semelparous life history, are primarily nocturnal, and rely on pheromone communication for reproduction; however, whether male investment in pheromone transport and release matches increases in spawning activity remains unknown. By measuring 1) 3keto-petromyzonol sulfate (3kPZS, a main pheromone component) and its biosynthetic precursor petromyzonol sulfate (PZS) in holding water and tissue samples at 6 points over the course of 24 hours, and 2) 3kPZS release over the course of several days, we demonstrate that 3kPZS release exhibits a consistent diel pattern across several days with elevated pheromone release just prior to sunset and at night. Trends in hepatic concentrations and circulatory transport of PZS and 3kPZS were consistent with patterns of 3kPZS release and suggest the possibility of direct upregulation in pheromone transport and release rather than observed release patterns being solely a byproduct of increased behavioral activity. Our results suggest males evolved a signaling strategy that synchronizes elevated pheromone release with nocturnal increases in sea lamprey behavior. This may be imperative to ensure that male signaling effort is not wasted in a species having a single, reproductive event.

Interior of sand fly (Diptera: Psychodidae) abdomen reveals novel structures involved in pheromone release: discovering the Manifold.

The males of many species of New World Phlebotomines produce volatile terpenoid chemicals which have been shown in Lutzomyia longipalpis s.l. and L. cruciata to be sex/aggregation pheromones which attract female and male conspecifics. Pheromone is produced in secretory cells surrounding a cuticular reservoir which collects the pheromone and passes it through a cuticular duct to the surface of the insect. On the surface the pheromone passes through a specialised structure prior to evaporation. The shape and distribution of the structures are highly diverse and differ according to species. They range in appearance from slightly raised domes (papules) to almost spherical apple shaped structures to slight depressions with central spikes and all with a central pore. They can occur either singly or in many hundreds distributed on most abdominal tergites or grouped on one. The pheromone secreting apparatus in sand flies and other insects have historically been examined from the exterior using scanning electron microscopy (SEM) and from the interior using transmission electron microscopy. In this study we used SEM to examine the interior cuticular structure of 3 members of the Lutzomyia longipalpis s.l. species complex and Migonemyia migonei and found a new structure associated with pheromone release which we have called the Manifold. The Manifold is a substantial structure siting in-line between the cuticular duct and the underside of the tergite. Differences in the size and shape of the Manifold may be related to the chemical structure of the pheromone. In addition to the importance of this hitherto unknown structure in the production, dissemination and ecology of the pheromone, as well as its potential taxonomic value, examination of the interior cuticle by SEM may help locate the secretory apparatus in important vector species where pheromonal activity has been inferred from behavioural studies but the external secretory structures or potential pheromones have not been found.

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.

Male accessory gland factors increased sex pheromone titres in the glands of Spodoptera litura female moths by inhibiting calling behavior

Moths are the most widely studied example of pheromones in animals. However, little is known about the mechanism of intrasexual and mating-related intersexual regulation of pheromone production and release in female moths. Our previous studies in Spodoptera litura found that mating induced a higher sex pheromone titre in the pheromone gland (PG) and mating or male accessory gland (MAG) extract suppressed female calling behavior. We therefore hypothesize that the inhibition of female calling behavior by mating or MAG factors likely suppresses the release of sex pheromones and thus results in a higher pheromone titre in the PG. To test this hypothesis, in the present study, we introduced an artificial calling behavior suppression treatment by gently knocking on and shaking the testing boxes contained moths once every 10 minutes. Results show that this treatment significantly increased pheromone titres in virgin or saline injected virgin females, and the increase rates are similar to those of mating and MAG extract treated ones. These results have suggested that the increase of sex pheromone titer in the female PG after mating in S. litura is due to the inhibition of female calling behavior by MAG factors. Moreover, results of this study also suggest that female calling behavior is positively correlated to pheromone release and likewise, the calling behavior and sex pheromone release in S. litura females are directly under the neural control, and modulated by molecular and environmental factors.