Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America.EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1^st-, 2^nd-, 3^rd-, 4^th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.Download video file.^{(130M, mp4)}     

Quantifying the Impact of Woodpecker Predation on Population Dynamics of the Emerald Ash Borer (Agrilus planipennis)

The emerald ash borer (EAB), Agrilus planipennis, is an invasive beetle that has killed millions of ash trees (Fraxinus spp.) since it was accidentally introduced to North America in the 1990s. Understanding how predators such as woodpeckers (Picidae) affect the population dynamics of EAB should enable us to more effectively manage the spread of this beetle, and toward this end we combined two experimental approaches to elucidate the relative importance of woodpecker predation on EAB populations. First, we examined wild populations of EAB in ash trees in New York, with each tree having a section screened to exclude woodpeckers. Second, we established experimental cohorts of EAB in ash trees in Maryland, and the cohorts on half of these trees were caged to exclude woodpeckers. The following spring these trees were debarked and the fates of the EAB larvae were determined. We found that trees from which woodpeckers were excluded consistently had significantly lower levels of predation, and that woodpecker predation comprised a greater source of mortality at sites with a more established wild infestation of EAB. Additionally, there was a considerable difference between New York and Maryland in the effect that woodpecker predation had on EAB population growth, suggesting that predation alone may not be a substantial factor in controlling EAB. In our experimental cohorts we also observed that trees from which woodpeckers were excluded had a significantly higher level of parasitism. The lower level of parasitism on EAB larvae found when exposed to woodpeckers has implications for EAB biological control, suggesting that it might be prudent to exclude woodpeckers from trees when attempting to establish parasitoid populations. Future studies may include utilizing EAB larval cohorts with a range of densities to explore the functional response of woodpeckers.     

The Role of Olfactory Cues in Short-Range Mate Finding by the Emerald Ash Borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae)

We investigated the relative importance of olfaction versus vision in the mate-finding behavior of Agrilus planipennis. When coupled in male–female, male–male and female–female pairs, attempts to mate occurred only in the male–female pairs, suggesting that beetles can identify the opposite sex before attempting to mate. In a set of sensory deprivation experiments with male–female pairs, we evaluated whether males could find females when deprived of their sense of olfaction, vision or both. Males whose antennae were blocked with model paint took significantly longer to find females and spent less time in copula compared to untreated males. Males whose eyes were similarly blocked did not differ in their mate finding capacity compared to untreated males. In a third experiment that compared both olfaction and vision, olfactorily impaired beetles never mated whereas the mate finding potential of visually impaired beetles did not differ from that of untreated beetles. Our results indicate that males can identify females before coming into physical contact with them, and that at short range (≤5 cm), volatile cues detected by olfaction are involved in mate finding by A. planipennis.     

Landing Surface Color Preferences of Spathius agrili (Hymenoptera: Braconidae), a Parasitoid of Emerald Ash Borer, Agrilus planipennis (Coleoptera: Buprestidae)

The color preferences for landing surfaces were examined for Spathius agrili (Hymenoptera: Braconidae), a parasitic wasp introduced for biocontrol of emerald ash borer, Agrilus planipennis (Coleoptera: Buprestidae). Lures with the 3-component pheromone blend of male S. agrili were used to activate upwind flight by virgin female S. agrili in a laminar flow wind tunnel. Paper discs with halves of two different colors (combination pairs of black, white, red, yellow, green, or purple), with the pheromone lure in the center, were tested to quantify preferences for landing on one color over another. Females landed preferentially on green, yellow, and white surfaces, and landed the least frequently on red, black, and purple surfaces. Changes in color preferences due to adjacent colors were observed and discussed.     

A new forest pest in Europe: a review of Emerald ash borer (Agrilus planipennis) invasion

In this publication, we review the biology, ecology, invasion history, impacts and management options of Emerald ash borer (EAB) Agrilus plannipennis, with a particular focus on its invasion in Europe. Agrilus planipennis (EAB) is a wood‐boring beetle native to East Asia. Having caused massive damages on ash species in North America in the last decades, it was first recorded in Europe in 2003 in Russia (Moscow). All ash (Fraxinus) species native to Europe and North America are known to be susceptible to EAB attacks, which cause high tree mortality even among formerly healthy trees. Recorded expansion rates are between 2.5 and 80 km/year in North America and between 13 and 41 km/year in European Russia. Given current expansion rates, EAB is expected to reach Central Europe within 15–20 years. A combination of mechanical, biological and chemical control and phytosanitary measures may reduce its impact, which nevertheless most likely will be substantial. There is an urgent need to identify native enemies in Europe, to test suitable biocontrol agents and to develop early detection and management measures. Although it is obvious that EAB will become a major pest in Europe, early and dedicated response will likely be able to reduce the level of ash mortality, and thus improve the opportunity for long‐term survival of ash as an important component in European forests.     

Core RNAi machinery and gene knockdown in the emerald ash borer (Agrilus planipennis)

The RNA interference (RNAi) technology has been widely used in insect functional genomics research and provides an alternative approach for insect pest management. To understand whether the emerald ash borer (Agrilus planipennis), an invasive and destructive coleopteran insect pest of ash tree (Fraxinus spp.), possesses a strong RNAi machinery that is capable of degrading target mRNA as a response to exogenous double-stranded RNA (dsRNA) induction, we identified three RNAi pathway core component genes, Dicer-2, Argonaute-2 and R2D2, from the A. planipennis genome sequence. Characterization of these core components revealed that they contain conserved domains essential for the proteins to function in the RNAi pathway. Phylogenetic analyses showed that they are closely related to homologs derived from other coleopteran species. We also delivered the dsRNA fragment of AplaScrB-2, a β-fructofuranosidase-encoding gene horizontally acquired by A. planipennis as we reported previously, into A. planipennis adults through microinjection. Quantitative real-time PCR analysis on the dsRNA-treated beetles demonstrated a significantly decreased gene expression level of AplaScrB-2 appearing on day 2 and lasting until at least day 6. This study is the first record of RNAi applied in A. planipennis.     

Novel visual-cue-based sticky traps for monitoring of emerald ash borers, Agrilus planipennis (Col., Buprestidae)

We examined various methods of trapping emerald ash borers (EAB), Agrilus planipennis Fairmaire, using solely visual cues based on previous work that has documented the importance of visual cues in EAB mate location. Here, we give the results of two of these methods, coloured sticky cards (yellow or blue), or live ash leaves covered with spray-on adhesive to which dead male EAB visual lures had been pinned. Feral male beetles were captured effectively on the sticky traps made from dead male EAB on ash leaves. These sticky-leaf-traps captured more male EAB when deployed in high-population density areas than low-density areas, but did capture EAB even at lower population densities. More feral males were captured on these traps when they were placed higher in the trees, regardless of the population density of EAB. Very few feral female EAB were captured using the sticky-leaf-traps. This novel method of EAB trapping may allow 'real-time' population detection and monitoring of EAB adults during the active flight period rather than locating larval galleries during the autumn and winter after adult flight and attack. Feral male beetles were also captured using standard yellow- or blue-coloured sticky cards to which male EAB had been affixed with adhesive; however, this type of trap was much less effective overall than using the sticky-leaf-traps. Furthermore, Agrilus cyanescens , a species similar in colour to EAB but smaller in size, showed a strong response to blue-coloured sticky traps to which dead male EAB had been affixed with adhesive, suggesting a general use of visual cues in the mating systems of some of the other Buprestidae as well.     

The overwintering physiology of the emerald ash borer, Agrilus planipennis fairmaire (coleoptera: buprestidae)

Ability to survive cold is an important factor in determining northern range limits of insects. The emerald ash borer (Agrilus planipennis) is an invasive beetle introduced from Asia that is causing extensive damage to ash trees in North America, but little is known about its cold tolerance. Herein, the cold tolerance strategy and mechanisms involved in the cold tolerance of the emerald ash borer were investigated, and seasonal changes in these mechanisms monitored. The majority of emerald ash borers survive winter as freeze-intolerant prepupae. In winter, A. planipennis prepupae have low supercooling points (∼−30 °C), which they achieve by accumulating high concentrations of glycerol (∼4 M) in their body fluids and by the synthesis of antifreeze agents. Cuticular waxes reduce inoculation from external ice. This is the first comprehensive study of seasonal changes in cold tolerance in a buprestid beetle.     

Development of a host-based semiochemical lure for trapping emerald ash borer Agrilus planipennis (Coleoptera: Buprestidae)

Bark volatiles from green ash Fraxinus pennsylvanica were tested for electrophysiological activity by Agrilus planipennis using gas chromatographic-electroantennographic detection (GC-EAD) and for behavioral activity using baited purple traps in Michigan. GC-EAD analysis of the headspace volatiles of bark tissue samples from 0- and 24-h-old fully girdled (stressed) ash trees showed that the latter had elevated sesquiterpene levels. Six of the elevated compounds consistently elicited antennal responses by both male and female A. planipennis. Five of the antennally active compounds were identified as alpha-cubebene, alpha-copaene, 7-epi-sesquithujene, trans-beta-caryophyllene, and alpha-humulene (alpha-caryophyllene). The sixth EAD-active compound remains unidentified. We monitored capture of adult A. planipennis on traps baited with several combinations of ash tree volatiles. Treatments included two natural oil distillates (Manuka and Phoebe oil) that were found to contain, respectively, high concentrations of four and five of the six antennally active ash bark volatiles. A four-component leaf lure developed by the USDA Forest Service and Canadian Forest Service was also tested. In three separate field studies, Manuka oil-baited traps caught significantly more adult beetles than unbaited traps. Lures designed to release 5, 50, and 500 mg of Manuka oil per day all caught more insects than unbaited traps. In a field test comparing and combining Phoebe oil with Manuka oil, Phoebe oil-baited traps caught significantly more beetles than either Manuka oil-baited traps or unbaited traps. We hypothesize that the improved attractancy of Phoebe oil to A. planipennis over Manuka oil is caused by the presence of the antennally active sesquiterpene, 7-epi-sesquithujene.     

Efficacy of trap and lure types for detection of Agrilus planipennis (Col., Buprestidae) at low density

Development of effective trapping tools for forest pests and evaluating the key components of these tools is necessary to locate early‐stage infestations and develop management responses to them. Agrilus planipennis Fairmaire (emerald ash borer) is an introduced pest of ash (Fraxinus spp. L.) in North America. The effectiveness of different trap and lure combinations were tested in areas with low and high density populations of A. planipennis. At low density sites, purple prism traps outperformed green traps and girdled ash trap trees in capture rates (adults per day) and rates of detection of A. planipennis. Also, manuka oil lures, used as a standard lure in a national survey programme, captured higher rates of A. planipennis than did previous standards of girdled ash trap trees. There was no logistic relationship between the detection of A. planipennis on a trap and the diameter of the ash tree from which the trap was suspended, possibly because of the use of artificial lures with these traps. There was also no difference in the mean number of A. lanipennis captured per day between ash species and between vigour rating of ash associated with the traps. However, traps placed in open grown and dominant trees captured more beetles than traps placed in lower canopy class trees. At sites defined as low and high density, there was no difference in the larval density per cm³ of phloem. This suggests that exposure time to A. planipennis has been shorter at those low density sites. By exploiting the trap and tree characteristics that improve A. planipennis capture rates and detection efficacy, there can be future improvement in management of this pest. If detection can occur before infested ash trees exhibit signs and symptoms, there may be a potential for reducing the mortality of ash within stands.     

Early detection of Agrilus planipennis: investigations into the attractive range of the sex pheromone (3Z)-lactone

The emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), is a highly destructive primary pest of ash (Fraxinus sp., Oleaceae) trees outside of its native range. Ash is an important component of many ecosystems and its loss would be detrimental to both the economy and the environment. The present study aimed to improve our understanding of the effectiveness of green sticky prism traps baited with host kairomone and insect pheromone lures for A. planipennis and to collect data for modelling the range of attraction of the pheromone (3Z)-dodecen-12-olide [(3Z)-lactone]. Traps were deployed over a single flight season in urban locations of Ontario, Canada, with low densities of EAB. Traps were placed in pairs of trees separated by not more than 25 m. All traps contained the host kairomone, (3Z)-hexenol, with the remaining half in each pairing additionally baited with (3Z)-lactone pheromone. Both lure types were highly effective in capturing EAB, with >90% detection rates overall. However, traps baited with the lactone pheromone and host volatile lures doubled trap captures of EAB over distances of at least 25 m from the nearest traps baited with only host volatiles. Although the baseline detection rate of traps containing (3Z)-hexenol alone is not significantly reduced compared with traps containing (3Z)-lactone, the overall trap effectiveness is significantly increased when (3Z)-lactone is present. The implications for the use of (3Z)-lactone at 3 mg per septum dose in an early warning trapping system are discussed. Trap layout methods and risk-based analysis models can now be further refined by including these data about the attractive range of lures and their behaviour in different plot environments.     

Factors affecting the survival of ash (Fraxinus spp.) trees infested by emerald ash borer (Agrilus planipennis)

Emerald ash borer (Agrilus planipennis) (EAB), an Asian woodboring beetle accidentally introduced in North America, has killed millions of ash (Fraxinus spp.) trees and is spreading rapidly. This study examined the effects of tree- and site-level factors on the mortality of ash trees in stands infested by EAB in OH, USA. Our data show that ash populations in forested sites can progress from healthy to almost complete mortality of mature trees within 6 years. Although the end result of nearly complete mortality does not vary, survival analysis with 5 years of data showed that some factors affected the rate of mortality. We found more rapid mortality in stands with lower densities of ash trees. This finding supports an extension of the resource dilution hypothesis whereby concentration of EAB on few trees in low ash density areas leads to rapid decline of these trees. This contradicts an extension of the resource concentration theory that greater host density increases relative pest abundance and host mortality. Although reductions in ash density via diversification may be desirable for other silvicultural, conservation, and management objectives in preparation for EAB, our study shows that the management strategy of reducing ash density is unlikely to protect the remaining ash trees. Survival analysis also showed that mortality was more rapid for trees shaded by other trees and for trees initially exhibiting dieback. In management scenarios where hazard tree removal must be spread over several years due to budget constraints, focusing initial tree removal on stressed trees is recommended.     

Modelling local and long-distance dispersal of invasive emerald ash borer Agrilus planipennis (Coleoptera) in North America

Limiting the damage by non-indigenous species requires rapid determination of current and potential distributions and vectors of dispersal, and development of appropriate management measures. The emerald ash borer ( Agrilus planipennis ), a wood-boring beetle native to South-East Asia, was first reported in the Great Lakes region during summer 2002. The beetle poses an enormous threat to native ash ( Fraxinus ) species of North America, as untreated trees in infested areas of Ontario, Michigan and Ohio suffer high mortality. We demonstrate that the borer has spread in North America through a combination of diffusive range extension, associated with local flights, and by long-distance 'jump' dispersal associated with human movement of infested sapling or contaminated firewood. Probability of infestation was inversely related to distance from borer epicentres but positively related to the size of human population centres. At least 9 of 39 populations that were first reported in Michigan during 2004 cannot be accounted for by local diffusion, raising the possibility that other unidentified mechanisms may be contributing to the dispersal of the beetle. In the absence of quarantine, by 2005 all of Michigan's lower peninsula was contained within the boundaries of potential diffusive range expansion. Infested ash saplings also were introduced from Michigan to Maryland during 2003, and subsequently transplanted to five sites in Maryland and Virginia. Quarantine and eradication measures have had mixed results: in the south-central USA, the species appears on the brink of eradication, whereas its distribution has continued to spread during 2005 in the Great Lakes region despite extensive containment and quarantine measures. Quarantine success in the Great Lakes region is encumbered by multiple dispersal vectors, larger borer population sizes and by the more extensive geographical distribution that was achieved prior to implementation of control measures.