UNL 2018 Improving Early Detection of The Emerald Ash Borer

Description

The Emerald Ash Borer (Agrilus planipennis) is one of the species of destructive tree beetles in North America. The beetle was initially identified in June 2002 in the southeast portion of the state of Michigan. As of October 2018, it is now found in 35 states, including Nebraska, and the Canadian provinces of Ontario, Quebec, New Brunswick, Nova Scotia and Manitoba (Emerald n.d.). A recent infestation has been confirmed in Lincoln as of August 2018. (Lincoln Journal Star, 2018). The following map shows the how widespread the EAB infestation is throughout North America.

Emerald Ash Borer Dispersion Map:

The Problem:

The emerald ash borer beetle is considered the most destructive forest pest ever seen in North America (McCullough & Usbourne, 2017). Emerald ash borers are highly invasive species native to Asia. They pose a threat to 16 species of native ash trees. Adult beetles are typically bright metallic green and about 8.5 millimeters (0.33 in) long.

The Emerald Ash Borer:

The emerald borer completes one generation every one or two years. Females lay eggs in outer layers of bark and in and crevices of ash trees (Fraxinae) from mid-June and late August. The eggs hatch in about two weeks and the larvae feed on the bark in an S-shaped pattern through the inner phloem and outer xylem. The larvae pupate and turn into adults typically during April and May. The adults appear in the late spring months and into early August and emerge from the tree in D-shaped exit holes. They later re-infest the old tree or find a new tree if the old tress is on the verge of dying (USDA, 2018). Visible signs of infestation only appear after their firm establishment.

Life Cycle of the Emerald Ash Borer:

Traps are triangular prisms that utilize vibrant green or purple colors, green leaf volatiles from ash trees, and other attractive compounds to lure the emerald ash borer. Many of these attractive compounds are present in a natural tree oil called Manuka oil, and for this reason this oil was often incorporated into EAB traps (Abell et al., 2015). These compounds are also known to be found in Phoebe oil in different proportions (Khrimian et. al, 2011). The six attractive compounds in these oils are α-cubebene, α-copaene, 7-epi-sesquithujene, trans-β-caryophyllene, α-humulene (α-caryophyllene), and eremophilene. Of these, it is thought that 7-epi-sesquithujene and eremophilene are the most attractive (Crook, 2010). It was discovered that purchasing either of these oils was very costly, in addition to the number of suppliers being sparse.

7-epi-Sesquithujene:

EAB Traps:

7-epi-Sesquithujene is a bicyclic sesquiterpene isolated from phoebe oil, an essential oil of the Brazilian walnut tree, Phoebe porosa. It is also produced by stressed ash trees and has been shown to elicit strong electrophysiological responses on emerald ash borer, Agrilus planipennis, antennae (Khrimian et. al, 2011). Research shows that sesquiterpenes can successfully be synthesized by both E. coli and the yeast Saccharomyces cerevisiae through the enzyme 7-epi-sesquithujene synthase (TPS4-B73). This biosynthesis requires the precursor farnesyl pyrophosphate (FPP) (Köllner et al, 2004). This chemical can be produced in E. coli by engineering the mevalonate pathway found in Saccharomyces cerevisiae and some enzymes from E. coli itself (Martin et. al, 2003). By implementing this pathway and the TPS4-B73 gene, E. coli can independently produce desirable levels of synthesis. After successful synthesis, 7-epi-sesquithujene can be purified, further analyzed, and used in emerald ash borer traps.

Our Approach:

There are a scarce number of approaches in dealing with emerald ash borer infestations. The current methods deal primarily with chemical treatment of ash trees within the vicinity of an already identified infestation. We believe that developing more effective traps is absolutely critical to slow down the rate at which the emerald ash borer is spreading by allowing people to treat their trees very early in the infestation or maybe before it even occurs. It has been shown that by the time the infestation is noticeable in the tree, it is already too late to treat the tree. One of the first indicators is the prevalence of woodpeckers surrounding the tree (Emerald Ash Borer Awareness, 2018). But this alone is not a good enough justification to promote invasive behavior on the tree, so the team sought a method that would promote a non-invasive approach to identifying infestations.

Life of a tree infested by Emerald Ash Borer:

A literature survey showed that it was impractical to chemically synthesize 7-epi-sesquithujene (Crook, 2010), our team decided to biosynthetically produce what appears to be an extremely valuable chemical. Biosynthesis of 7-epi-sesquithujene requires a long biosynthetic route that utilizes several well-studied biochemical pathways. The first pathway entails the production of mevalonate from acetyl-CoA. This conversion requires three different enzymes: acetyl-CoA acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), which is the rate-limiting step of the pathway. This mevalonate pathway is encoded by plasmid 100167, which we obtained from Addgene.

Mevalonate Pathway Plasmid Map:

The mevalonate is then converted to farnesyl pyrophosphate via the mevalonate-based isoprenoid synthesis (MBIS) pathway, which requires the activities of five different enzymes. This pathway is also encoded on the plasmid 100167. Previous report showed that the overexpression of the mevalonate kinase was beneficial to the biosynthesis, since the it is a rate limiting enzyme (Anthony et. al, 2009). We obtained plasmid 100169 from Addgene, which contains a second copy of the mevalonate kinase-encoding gene. Our project focused on the biosynthetic approach to produce 7-epi-sesquithujene and are looking forward to further experimentation for the possibility of scaling up.

Mevalonate Pathway:

Works Cited

USDA. Emerald Ash Borer (Agrilus planipennis). https://www.invasivespeciesinfo.gov/animals/eab.shtml (accessed Oct 12, 2018).
Lincoln Journal Star. Emerald Ash Borer in Lincoln https://journalstar.com/lifestyles/home-and-garden/emerald-ash-borer-confirmed-in-lincoln/article_fb88ba88-7c13-5d30-9df7-659bf36a05e1.html (accessed Oct 12, 2018).
USDA. Emerald Ash Borer (Agrilus planipennis). https://www.nrs.fs.fed.us/disturbance/invasive_species/eab/biology_ecology/planipennis/ (accessed Oct 12, 2018).
Trapping Techniques for Emerald Ash Borer and Its Introduced Parasitoids, Abell et al., 2015, p. 113 https://www.fs.fed.us/nrs/pubs/jrnl/2015/nrs_2015_abell_001.pdf (accessed Oct 12, 2018).
McCullough, D.; Usbourne, R. Emerald Ash Borer Information Network. http://emeraldashborer.info/ (accessed Oct 13, 2018).
Khrimian, A.; Cossé, A. A.; Crook, D. J. Absolute configuration of 7-epi-sesquithujene. https://www.ncbi.nlm.nih.gov/pubmed/21574561 (accessed Oct 13, 2018).
USDA. The cost of the emerald ash borer infestation. https://www.nrs.fs.fed.us/disturbance/invasive_species/eab/effects_impacts/cost_of_infestation/ (accessed Oct 13, 2018).
Anthony, J. R.; Anthony, L. C.; Nowroozi, F.; Kwon, G.; Newman, J. D.; Keasling, J. D. Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. https://www.sciencedirect.com/science/article/pii/S1096717608000438?via=ihub (accessed Oct 12, 2018).

Image Citations:

Emerald Ash Borer Information Network. http://www.emeraldashborer.info/ (accessed Oct 13, 2018). (Image 1)
Emerald Ash Borer detected in South Carolina. https://www.state.sc.us/forest/emeraldashborerdetected.htm (accessed Oct 13, 2018). (Image 2)
Hoppe, A. Insects, Diseases, And Plant Health Care. https://www.hoppetreeservice.com/index.php/en/tree-care-info/insects,-diseases,-and-plant-health-care/190-emerald-ash-borer-–-now-is-the-time-to-act (accessed Oct 13, 2018). (Image 3)
7-epi-sesquithujene. http://www.chemspider.com/Chemical-Structure.26606391.html (accessed Oct 13, 2018). (Image 4)
Trapping and Attractants. https://www.nrs.fs.fed.us/disturbance/invasive_species/eab/risk_detection_spread/trapping_attractants/ (accessed Oct 14, 2018). (Image 5)
Anthony, J. R.; Anthony, L. C.; Nowroozi, F.; Kwon, G.; Newman, J. D.; Keasling, J. D. Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. https://www.sciencedirect.com/science/article/pii/S1096717608000438?via=ihub (accessed Oct 12, 2018). (Image 6)

Team:UNebraska-Lincoln/Description