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 DNA Melt Curve Analysis for the Identification of Lepidopteran Pests in Almonds and Pistachios

Almonds and pistachios are fed upon by a diverse assemblage of lepidopteran insects, several of which are economically important pests. Unfortunately, identification of these pests can be difficult, as specimens are frequently damaged during collection, occur in traps with non-target species, and are morphologically similar up to their third instar. Here, we present a quantitative PCR based melt curve analysis for simple, rapid, and accurate identification of six lepidopteran pests of almonds and pistachios: navel orangeworm (Amyelois transitella), peach twig borer (Anarsia lineatella), oriental fruit moth (Grapholita molesta), obliquebanded leafroller (Choristoneura rosaceana), raisin moth (Cadra figulilella), and Indian meal moth (Plodia interpunctella). In this approach, the dissociation (melt) temperature(s) of a 658 bp section of cytochrome c oxidase subunit 1 was determined using quantitative PCR (qPCR). Within these six species, the distribution and the number of melt peak temperatures provide an unambiguous species level identification that is reproducible when unsheared DNA can be extracted. The test is robust across a variety of sampling approaches including insects removed from sticky card traps, museum specimens, and samples that were left in the field for up to 7 days. The melt curve’s simplicity allows it to be performed in any basic molecular biology laboratory with a quantitative PCR.

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.

Individual and Additive Effects of Insecticide and Mating Disruption in Integrated Management of Navel Orangeworm in Almonds

Damage from Amyelois transitella, a key pest of almonds in California, is managed by destruction of overwintering hosts, timely harvest, and insecticides. Mating disruption has been an increasingly frequent addition to these management tools. Efficacy of mating disruption for control of navel orangeworm damage has been demonstrated in experiments that included control plots not treated with either mating disruption or insecticide. However, the navel orangeworm flies much farther than many orchard pests, so large plots of an expensive crop are required for such research. A large almond orchard was subdivided into replicate blocks of 96 to 224 ha and used to compare harvest damage from navel orangeworm in almonds treated with both mating disruption and insecticide, or with either alone. Regression of navel orangeworm damage in researcher-collected harvest samples from the interior and center of management blocks on damage in huller samples found good correlation for both and supported previous assumptions that huller samples underreport navel orangeworm damage. Blocks treated with both mating disruption and insecticide had lower damage than those treated with either alone in 9 of the 10 years examined. Use of insecticide had a stronger impact than doubling the dispenser rate from 2.5 to 5 per ha, and long-term comparisons of relative navel orangeworm damage to earlier- and later-harvested varieties revealed greater variation than previously demonstrated. These findings are an economically important confirmation of trade-offs in economic management of this critical pest. Additional monitoring tools and research tactics will be necessary to fulfill the potential of mating disruption to reduce insecticide use for navel orangeworm.

Differential regulation of cytochrome P450 genes associated with biosynthesis and detoxification in bifenthrin-resistant populations of navel orangewom (Amyelois transitella)

Pyrethroid resistance was first reported in 2013 for the navel orangeworm, Amyelois transitella, but the genetic underpinnings of pyrethroid resistance are unknown. We investigated the role of cytochrome P450 monooxygenases (P450s) belonging to the CYP3 and CYP4 clans using colonies derived from individuals collected in 2016 from almond orchards in two counties. One colony (ALM) originated from an almond orchard in Madera County with no reported pyrethroid resistance and the second colony (R347) originated from the same Kern County orchard where pyrethroid resistance was first reported. We used high-throughput quantitative real-time PCR (qRT-PCR) analyses of 65 P450s in the CYP3 and CYP4 clans of A. transitella to identify P450s induced by bifenthrin and associated with pyrethroid resistance. Nine P450s were constitutively overexpressed in R347 compared to ALM, including CYP6AE54 (11.7-fold), belonging to a subfamily associated with metabolic pesticide detoxification in Lepidoptera and CYP4G89 (33-fold) belonging to a subfamily associated with cuticular hydrocarbon (CHC) synthesis and resistance via reduced pesticide penetrance. Cuticular hydrocarbons analysis revealed that R347 produced twice as many total CHCs in the egg and adult stages as ALM. Topical toxicity bioassays for R347 determined that egg mortality was reduced at low bifenthrin concentrations and larval mortality was reduced at high concentrations of bifenthrin compared to ALM. Our discovery of both changes in metabolism and production of CHCs for R347 have implications for the possible decreased efficacy of other classes of insecticide used to control this insect. The threat of widespread pyrethroid resistance combined with the potential for cross-resistance to develop through the mechanism of reduced penetrance warrants developing management strategies that facilitate insecticide passage across the cuticle.

Management of Navel Orangeworm (Lepidoptera: Pyralidae) Using Four Commercial Mating Disruption Systems in California Almonds

The navel orangeworm, Amyelois transitella (Walker), is the most significant pest of California almonds. Direct feeding on the kernel by the larvae causes reductions in salable crop, crop quality, and exportability. Pheromone mating disruption (MD) targeting navel orangeworm is a relatively new technique with the potential to improve management. In 2017, we used replicated ~16-ha plots to compare the efficacy of four commercial MD systems (CheckMate, Cidetrak, Isomate, and Semios) for their relative impacts on the number of navel orangeworm in monitoring traps and crop quality. From 2017 to 2018, we conducted nine direct comparison studies in 16 to 40 ha almond orchards to compare conventional pest management programs to programs incorporating pheromone MD systems. Across all studies, MD reduced male moth captures in pheromone traps by >94%. In the efficacy study, use of mating disruption led to 35% and 53% reductions in kernel damage in Nonpareil and pollinizer cultivars, respectively, and an average increase in crop value of $370 ha−1. In the direct comparison, kernel damage to Nonpareil and pollinizer cultivars was reduced by 65% and 78%, respectively, resulting in an average increase in crop value of $357 ha−1. Economic analyses showed that increases in crop returns exceeded the costs of implementing MD systems with the break-even point ranging from 0.86 to 1.06% of kernel damage. These results suggest that adding MD to an existing navel orangeworm management program is a cost-effective way to reduce damage while promoting sustainable pest management practices.

Selective sweeps in a nutshell: the genomic footprint of rapid insecticide resistance evolution in the almond agroecosystem

Among the most familiar forms of human-driven evolution on ecological time scales is the rapid acquisition of resistance to pesticides by insects. Since the widespread adoption of synthetic organic insecticides in the mid-twentieth century, over 500 arthropod species have evolved resistance to at least one insecticide. Efforts to determine the genetic bases of insecticide resistance have historically focused on individual loci, but the availability of genomic tools has facilitated the screening of genome-wide characteristics. We re-sequenced three contemporary populations of the navel orangeworm (Amyelois transitella), the principal pest of almond orchards in California, differing in bifenthrin resistance status to examine insecticide-induced changes in the population genomic landscape of this species. We detected an exceptionally large region with virtually no polymorphisms, extending to up to 1.3 Mb in the resistant population. This selective sweep includes genes associated with pyrethroid and DDT resistance, including a cytochrome P450 gene cluster and the gene encoding the voltage-gated sodium channel para. Moreover, the sequence along the sweep is nearly identical in the genome assembled from a population founded in 1966, suggesting that the foundation for insecticide resistance may date back a half-century, when California’s Central Valley experienced massive area-wide applications of DDT for pest control.

Evaluating Response of Mass-Reared and Irradiated Navel Orangeworm, Amyelois transitella (Lepidoptera: Pyralidae), to Crude Female Pheromone Extract

The navel orangeworm, Amyleois transitella (Lepidoptera: Pyralidae), is a key pest of almonds and pistachios in California. Larvae directly feed on nuts, reducing quality and yield, and adults can introduce fungi that produce aflatoxins. The development of sterile insect technique (SIT) is currently being explored as a management tool for this pest. Large quantities of A. transitella are mass-reared, irradiated, and shipped to California from a USDA APHIS facility in Phoenix, AZ. Preliminary field releases of sterile A. transitella from this facility resulted in poor recovery of males in pheromone traps, raising concerns that mass-reared male A. transitella may not be responding to pheromone from virgin females. In this study, a wind tunnel was used to evaluate the response of both irradiated and non-irradiated mass-reared A. transitella males to crude pheromone extract from females, and their performance was compared to two strains of locally reared non-irradiated A. transitella. While initial responses associated with pheromone detection where similar between mass-reared and locally reared moths, a lower proportion of the mass-reared moths ultimately made contact with the pheromone source. Surprisingly, the addition of irradiation did not further decrease their performance. While mass-reared moths respond to pheromone, their ability to locate and make contact with the pheromone source appears to be impeded. The underlying mechanism remains unclear, but is likely related to damage incurred during the mass-rearing and shipping process.