Name: building double-decker traps for early detection of emerald ash borer
Uploaded: 2017-10-04T11:30:00
Description: Watch this Scientific Journal Video about Building Double-decker Traps for Early Detection of Emerald Ash Borer at JoVE.com

Several technicians and graduate students at Michigan State University have helped to develop, refine and evaluate the DD trap design over the years, including Andrea Anulewicz, Robert McDonald and Nathan Siegert. We thank James Wieferich and Jeremy Lowell (MSU) for their assistance in developing the instructions for DD installation. James Wieferich and Molly Robinett (MSU) reviewed an earlier draft of this manuscript and we appreciate their suggestions. Joseph Francese and Damon Crook (USDA APHIS) generously shared their observations on EAB response to color and host volatiles. Funding for DD trap development and evaluation was provided by grants from the USDA Forest Service, Northeastern Area, Forest Health Protection.

1. Prepare the Green and Purple Panels
<ol>
	<li>Acquire green and purple corrugated plastic panels (120 cm &#215; 60 cm) for EAB trapping from a commercial distributor of pest management supplies. Use a box-cutter or utility knife to score two fold lines in each panel by partially cutting through the plastic along the vertical corrugations, 40 cm from the two short edges of the panel. This allows the panel to be folded into a 3-sided prism (each face will be 40 x 60 cm). Panels are usually scored by the distributor to...

Both the design and placement of DD traps exploit the attraction of adult EAB beetles to specific shades of color and to light. The green prism on the top of the PVC pipe is most attractive to male beetles, who spend their life span feeding on ash leaves, as well as mating32,36,38,39. The lower purple prism ensures traps are also attractive to female beetles32. Like male...

The emerald ash borer (EAB) (Agrilus planipennis Fairmaire) (Coleoptera: Buprestidae) has killed hundreds of millions of ash (Fraxinus spp.) trees since it was first identified in 2002 in the greater Detroit, Michigan metropolitan area and in nearby Windsor, Ontario, Canada. Inadvertent spread of infested ash trees, logs and firewood, along with natural dispersal of beetles, have resulted in EAB establishment in at least 27 states, and two Canadian provinces to date1. Recent reports indicate the EAB has also invaded Moscow, Russia, where it is killing landscape ash trees2,3, generating additional concern about its potential spread into Europe. Interspecific variation in EAB host preference and resistance among North American ash species has been documented4,5,6,7,8,9, but virtually all ash species in North America are likely to be suitable hosts. Catastrophic levels of ash mortality have been recorded in areas of Michigan and Ohio10,11,12, with associated ecological and economic impacts13,14,15,16.
Effective methods to detect new EAB infestations and monitor low density populations are critical aspects of managing this invasive pest in urban, residential and forested settings. Early detection provides time to develop a strategy, secure funding and implement activities to reduce impacts of the EAB. For example, municipal officials and homeowners can begin treating valuable ash in landscapes with systemic insecticides before damage caused by increasing larval densities limits efficacy of these products17,18. Similarly, identification of a new infestation and reliable information about local EAB distribution gives foresters and property owners the opportunity to implement timber sales, species conversion or other activities to reduce economic costs or ecological effects of ash mortality.
Early detection, delimitation, and effective monitoring of low-density EAB populations, however, remain difficult. Visual surveys to identify newly infested ash trees are not reliable because ash rarely exhibit external signs or symptoms of EAB infestation until larval densities build to moderate or even high levels4,19. The most effective means of detecting very low densities of EAB involve using girdled ash detection trees19,20,21,22. Ash trees are girdled in spring or early summer by removing a band of outer bark and phloem around the circumference of the trunk, which stresses trees, increasing their attraction to adult EAB beetles. Girdled trees can be debarked in autumn or winter to identify EAB larval presence and density. Although girdled ash trees have been used operationally for EAB detection19,23,24,25, they are problematic. Debarking girdled trees can be labor intensive and locating suitable trees for girdling can be difficult, especially in urban or residential areas or when surveys must be conducted for multiple years19.
Artificial traps baited with EAB attractants eliminate many concerns associated with using girdled ash detection trees. In contrast to other important forest pests such as gypsy moth (Lymantria dispar L) and some Scolytinae bark beetles that produce long distance sex or aggregation pheromones, to date, no effective long-distance pheromones have been found for EAB. A short range sex pheromone, cis-lactone, may facilitate mating26,27, but in field trials, cis-lactone lures have not consistently increased EAB attraction to artificial traps28. Adult beetles rely on volatile compounds emitted by ash leaves, bark and wood to identify their host trees29,30,31 and encounter potential mates. Several volatile compounds have been evaluated for use in lures to attract adult EABs to artificial traps27,32. Currently, traps used operationally for EAB detection surveys in the U.S. are baited with lures containing cis-3-hexenol, a common green leaf volatile produced by ash foliage30,33. In previous years, EAB traps used for U.S. surveys have also been baited with Manuka oil, which is extracted from the New Zealand tea tree (Leptospermum scoparium Forst and Forst ) or Phoebe oil, an extract of the Brazilian walnut tree (Phoebe porosa Mez.); both contain several sesquiterpenes that are also present in ash bark29. Problems with inconsistent supplies of these natural oils, however, have restricted their use.
In addition to host-produced volatiles, adult EAB beetles respond to visual stimuli, including color and light20,32,34,35. Early studies showed EAB adults, which are relatively agile fliers, were rarely captured by black funnel traps baited with various ash volatiles (DGM and TMP, unpublished data). Other trap designs, such as cross-vane traps, were evaluated but the aversion of EAB beetles to dark spaces and shadows limited the effectiveness of these traps.
Development of a three-sided prism, which can be coated with clear insect trapping glue35 to capture beetles, was a substantial improvement in trap design. Attraction of adult EABs to color has also been extensively evaluated in field trials and in laboratory studies with a retinograph34. Results show EAB beetles are consistently attracted to specific shades of green and purple32,36. Prism traps fabricated from colored corrugated plastic are now widely used in EAB survey activities in the U.S. and Canada.
Because EAB adults are strongly attracted to light, beetles are much more likely to colonize open-grown trees than shaded trees20,21. Guidelines for EAB detection surveys in the U.S. required individual prism traps to be suspended from a mid-canopy branch in an ash tree growing along a road or the edge of a wooded area37. In theory, this should ensure that at least one panel of the prism is exposed to sunlight. Operationally, however, prism traps may be partially shadowed by overhead branches or by adjacent or nearby trees. Sticky panel surfaces are frequently blown into foliage, resulting in leaves adhering to and obscuring at least a portion of one or more panels.
Double-decker (DD) traps were developed to integrate multiple visual and olfactory cues to enhance attraction of EAB beetles. Each DD trap is comprised of one green and one purple corrugated plastic prism attached to a 3 m tall schedule 40 polyvinyl chloride (PVC) pipe (10 cm diameter), which is supported by sliding the PVC pipe over a t-post. Using both green and purple prisms is designed to attract both sexes of EAB beetles32,36,38,39. Additionally, rather than being suspended from a branch in the canopy of an ash tree, the DD traps are placed in full sun, 5-10 m away from ash trees along the edge of a wooded area or in the midst of scattered, open-grown ash trees.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Light green corrugated plastic panel: 120 cm x 60 cm</td>
			<td>Great Lakes IPM; www.greatlakesipm.com</td>
			<td>IPM-EAB GR</td>
			<td colspan="13" rowspan="3">All three surfaces of each prism need to be covered with clear insect trapping glue, even if the panels are pre-glued. Pre-glued panels are often not sticky enough to consistently capture or retain EAB beetles.&#160; Other clear insect trapping glue products are available but are considerably more difficult to apply.&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Light purple corrugated plastic panel: 120 cm x 60 cm</td>
			<td>Great Lakes IPM; www.greatlakesipm.com</td>
			<td>IPM-EAB&#160; LP</td>
		</tr>
		<tr>
			<td>Large cable tie (4): 60 cm with a 79 kg capacity</td>
			<td>Cabletiesandmore.com; http://www.cabletiesandmore.com/cableties.php</td>
			<td>CT-24-NU-100PK</td>
		</tr>
		<tr>
			<td>Medium cable ties (4): 20 cm with a 22.7 kg capacity</td>
			<td>Cabletiesandmore.com; http://www.cabletiesandmore.com/cableties.php</td>
			<td>CT261</td>
			<td colspan="13" rowspan="1"></td>
		</tr>
		<tr>
			<td>Small cable tie: 10 cm with a 8.2 kg capacity</td>
			<td>Cabletiesandmore.com; http://www.cabletiesandmore.com/cableties.php</td>
			<td>CT204</td>
			<td colspan="13" rowspan="1"></td>
		</tr>
		<tr>
			<td>cis-3-hexanol pouch</td>
			<td>Synergy Semiochemicals; http://www.semiochemical.com/html/buprestids.html)</td>
			<td>3136</td>
			<td colspan="13" rowspan="4">Lures used to bait DD traps consist of pouches containing cis-3-hexenol, a non-toxic compound present in ash leaves.&#160; One pouch is attached to the lower edge of the top prism using a small cable tie.&#160; Each pouch of cis-3-hexenol has a release rate of approximately 50 mg/day. Note that cis-3-hexenol is sometimes written as Z-3-hexenol.&#160;</td>
		</tr>
		<tr>
			<td>Aphinity Hexenol</td>
			<td>Sylvar Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Lure GLV4 emerald ash borer</td>
			<td>Chemtica, Heredia, Costa Rica</td>
			<td></td>
		</tr>
		<tr>
			<td>cis-3-hexanol pouch</td>
			<td>WestGreen Global Technologies; http://www.westgreenglobaltechnologies.com/</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear insect trapping glue&#160;</td>
			<td>Hummert International; http://www.hummert.com/product-details/8196/pestick</td>
			<td>01-3522-1&#160;&#160;</td>
			<td colspan="13" rowspan="1"></td>
		</tr>
		<tr>
			<td>Histoclear II histological clearing agent</td>
			<td>National Diagnostics; www.nationaldiagnostics.com</td>
			<td>HS-202</td>
			<td colspan="13">Histoclear II will be needed to remove the sticky insect glue from suspect beetles.&#160; Other histological clearing agents are available but may not remove the glue and some products dissolve plastic, an important consideration if plastic containers are used for soaking the beetles.&#160;</td>
		</tr>
		<tr>
			<td>Histoclear II histological clearing agent</td>
			<td>Great Lakes IPM; www.greatlakesipm.com</td>
			<td>10011</td>
			<td colspan="13">Histoclear II will be needed to remove the sticky insect glue from suspect beetles.&#160; Other histological clearing agents are available but may not remove the glue and some products dissolve plastic, an important consideration if plastic containers are used for soaking the beetles.&#160;</td>
		</tr>
		<tr>
			<td>t-post: 1.5 m</td>
			<td>multiple sources</td>
			<td></td>
			<td colspan="13" rowspan="1">A t-post (5 feet tall) (1.5 m) is used to support the PVC pipe.&#160;&#160;</td>
		</tr>
		<tr>
			<td>post pounder</td>
			<td>multiple sources</td>
			<td></td>
			<td colspan="13" rowspan="1">Use a post pounder to set t-posts into the ground. No additional support is necessary.</td>
		</tr>
		<tr>
			<td>HDPE (high density polyethylene) PVC pipe : 3 m x 10 cm diameter</td>
			<td>multiple sources</td>
			<td colspan="14" rowspan="1"></td>
		</tr>
		<tr>
			<td>Forceps (rigid)</td>
			<td>multiple sources</td>
			<td></td>
			<td colspan="13">Forceps (tweezers) will be needed to remove suspect beetles from the traps. Rigid forceps work better than flexible forceps.&#160;</td>
		</tr>
		<tr>
			<td>Latex gloves</td>
			<td>multiple sources</td>
			<td></td>
			<td colspan="13">Latex gloves are needed for applying the insect trapping glue to the prisms and for checking the traps to collect EAB beetles.&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Baby oil or baby wipes&#160;</td>
			<td>multiple sources</td>
			<td></td>
			<td colspan="13" rowspan="1">Baby oil or baby wipes are helpful for removing the trapping glue from hands and equipment.&#160;</td>
		</tr>
		<tr>
			<td>Re-sealable plastic specimen bags: 5 cm x 8 cm&#160;</td>
			<td>multiple sources</td>
			<td></td>
			<td colspan="13">Small re-sealable plastic specimen bags are useful for collecting beetles from traps.&#160; Each bag should be labelled, either with pre-made, adhesive labels or with soft felt pens.&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td colspan="3">Guides to help with distinguishing EAB from beetles native to North America are available on the national EAB website at www.emeraldashborer.info. &#160;</td>
			<td colspan="13" rowspan="1"></td>
		</tr>
	</tbody>
</table>

building double-decker traps for early detection of emerald ash borer

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions of forest and landscape ash (Fraxinus spp.) trees. Several artificial trap designs to attract and capture EAB beetles have been developed to detect, delineate, and monitor infestations. Double-decker (DD) traps consist of two corrugated plastic prisms, one green and one purple, attached to a 3 m tall polyvinyl chloride (PVC) pipe supported by a t-post. The green prism at the top of the PVC pipe is baited with cis-3-hexenol, a compound produced by ash foliage. Surfaces of both prisms are coated with sticky insect glue to capture adult EAB beetles. Double-decker traps should be placed near ash trees but in open areas, exposed to sun. Double-decker trap construction and placement are presented here, along with a summary of field experiments demonstrating the efficacy of DD traps in capturing EAB beetles. In a recent study in sites with relatively low EAB densities, double-decker traps captured significantly more EAB than green or purple prism traps or green funnel traps, all of which are designed to be suspended from a branch in the canopy of ash trees. A greater percentage of double decker traps were positive, i.e., captured at least one EAB, than the prism traps or funnel traps that were hung in ash tree canopies.

In a large scale study, three artificial trap designs as well as girdled ash trees were deployed systematically, 10-20 m apart, across a newly infested 16 ha forested area with a very low density of EAB25. Artificial trap designs tested included purple prisms baited with Manuka oil and suspended &#62;3 m high from a branch in the canopy of ash trees, 3 m tall DD traps with two green panels, and DD traps with two purple panels supported by t-posts. Double-decker tra...

Watch this Scientific Journal Video about Building Double-decker Traps for Early Detection of Emerald Ash Borer at JoVE.com

Building Double-decker Traps for Early Detection of Emerald Ash Borer

Effective traps to attract and capture the emerald ash borer (EAB) are a key element of detecting and managing this invasive pest. Double-decker traps, placed in full sun near ash trees, incorporate visual and olfactory cues and were more likely to capture EAB than other trap designs in field trials.

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions ...

Our goal is do demonstrate how to build, set up, and monitor double-decker traps to detect or monitor low densities of Emerald Ash Borer. Baited double-decker traps, if correctly assembled and positioned, are consistently more effective than any artificial traps at capturing adult Emerald Ash Borer beetles in low density populations. We have conducted Emerald Ash Borer trapping studies in more than 50 locations over the past 8 years.And this remains the most effective artificial trap design we have tested. Demonstrating the procedure today will be James Wieferich and Andrew Tluczek, both of whom are research technicians in my laboratory. To begin, using a half inch bit, drill a pair of holes into each scored plastic panel, four inches from the top of the panel, and four inches from either side of the left fold.Next prepare a three meter long, 10 centimeter in diameter, PVC pipe. Using a half inch bit, drill a pair of holes four inches from the top of the PVC pipe and 3.5 inches apart. Align the holes horizontally.Then drill a second pair of holes, 3.5 inches apart, 52 inches below the top of the pipe, and directly in line with the first set of holes. Now install a T Post at the desired location using a post pounder. Drive the post deep enough so that the flange is just below the soil surface, but there is no need to go any deeper.Guide wires, or additional supports, are not necessary. Next, fold the plastic panels into thirds. Then feed the two small flaps through the slits.And use two eight inch cable ties to secure the edges of the panel together, forming a three sided prism. Next, slide the prism onto the PVC pipe and down to the lower set of holes. Then feed a two foot long cable tie through the top left hole of the prism, through the holes on the PVC pipe, and out through the hole on the right side of the prism.Then close and tighten the cable tie, making sure the cable tie is not twisted. Before proceeding, make sure the prism is tight against the pipe between the holes. Now feed another long cable tie through the bottom hole of the prism, around the pipe, and through the corresponding hole on the adjacent side of the prism.Tighten this tie like the other. The next step is to attach the green prism to the pipe at the upper set of holes, using the same technique used to attach the purple prism. To apply insect trapping glue to a trap, set the base of the PVC pipe on the ground and prop the inner face of the upper prism on the T Post.To bait the trap with the lure, look for a small hole near the bottom edge of a panel on the green prism. Then using a four inch cable tie, secure a pouch of SIS 3-Hexanol to the lower edge of the green prism. This lure should last 10 weeks or longer.Wearing disposable gloves, scoop up two to three handfuls of glue and smear it across the surface of the panel. Thoroughly coat the panel on all three sides, even pre-glued panels often require additional glue to capture EAB beetles. Check traps a two to four week intervals if possible to avoid losing captured beetles which occasionally drop off the traps, especially if heavy rain occurs.Examine all three sides of the lower prism for EAB beetles. Then, using disposable latex gloves, lift the PVC pipe straight up and off the T Post, and prop the inner face of the prism on the T Post, exposing one panel of both prisms. Then, take the trap off the T Post, prop it on the T Post, and check the upper green prism.Use forceps to remove any suspect EAB beetles from the prisms and place them in a resealable plastic specimen bag. Flies, Click beetles, and various other insects will inevitably be captured on the traps. It takes a keen eye to spot a EAB beetle.Rotate the pipe to examine the other panels. Once the trap is fully checked, slide the pipe back over the T Post. Later, soak the possible EAB beetles in a non-toxic histological clearing agent for a day or two to remove the insect trapping glue.Then, confirm their identity under a dissecting microscope. Overall, the double-decker traps were the most effective at catching EAB beetles. In follow up experiments, the green purple combination proved more effective than the purple purple combination.After watching this video, you should have a good understanding of how to set up and check double-decker traps to detect and monitor low density populations of Emerald Ash Borer. These traps are an important aspect of managing Emerald Ash Borer. Early detection provides time to develop and implement effective control and management strategies for this invasive pest.

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Research

JoVE Journal

Environment

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps&#160;and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter&#160;scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,&#160;(2) superior optical sectioning ability,&#160;and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Comprehensive monitoring of coffee berry borer and host plant dynamics is essential for aggregating landscape-level data to improve management of this invasive pest. Here, we present a protocol for scientific monitoring of coffee berry borer movement, infestation, mortality, coffee plant phenology, weather, and farm management via a mobile electronic data recording application.

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

Fluorescent Paper Strips for the Detection of Diesel Adulteration with Smartphone Read-out

Three fluorescent molecular rotors of 4-dimethylamino-4-nitrostilbene (4-DNS) were investigated for their potential use as viscosity probes to indicate the content of kerosene in diesel/kerosene blends, a wide-spread activity to adulterate fuel. In solvents with low viscosity, the dyes rapidly deactivate via a so-called twisted intramolecular charge transfer state, efficiently quenching the fluorescence. Measurements of diesel/kerosene blends revealed a good linear correlation between the decrease in fluorescence and the increase of the fraction of the less viscous kerosene in diesel/kerosene blends. Immobilization of the hydroxy derivative 4-DNS-OH in cellulose paper yielded test strips that preserve the fluorescent indicator&#39;s behavior. Combination of the strips with a reader based on a smartphone and a controlling app allowed to create a simple field test. The method can reliably detect the presence of kerosene in diesel from 7 to 100%, outperforming present standard methods for diesel adulteration.

Here, we present a protocol to detect the adulteration of diesel with kerosene using test strips coated with a fluorescent viscosity probe together with a smartphone-based analysis system.

Three fluorescent molecular rotors of 4-dimethylamino-4-nitrostilbene (4-DNS) were investigated for their potential use as viscosity probes to indicate ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to bacteria control the amount that will accumulate in staple food crops. The bioavailability of these metals is dependent on their speciation in solution, but more particularly under anoxic conditions where Hg is methylated to toxic monomethylmercury (MeHg) and Cd persists in the rhizosphere. Whole-cell microbial biosensors give a quantifiable signal when a metal enters the cytosol and therefore are useful tools to assess metal bioavailability. Unfortunately, most biosensing efforts have so far been constrained to oxic environments due to the limited ability of existing reporter proteins to function in the absence of oxygen. In this study, we present our effort to develop and optimize a whole-cell biosensor assay capable of functioning anaerobically that can detect metals under anoxic condition in quasi-real time and within hours. We describe how the biosensor can help assess how chemical variables relevant to the environmental cycling of metals affect their bioavailability. The following protocol includes methods to (1) prepare Hg and Cd standards under anoxic conditions, (2) prepare the biosensor in the absence of oxygen, (3) design and execute an experiment to determine how a series of variable affects Hg or Cd bioavailability, and (4) to quantify and interpret biosensor data. We show the linear ranges of the biosensors and provide examples showing the method's ability to distinguish between metal bioavailability and toxicity by utilizing both metal-inducible and constitutive strains.

Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to ...

A Precise and Autonomous System for the Detection of Insect Emergence Patterns

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of emerging insects they can detect. In order to obtain precise measurement of insect emergence, it is necessary for systems to be semi-automated and able to measure large numbers of emerging insects. We addressed these issues by designing and building a system that is automated and can measure emergence of up to 1200 insects. We modified the existing &#34;falling-ball&#34; system using Arduino microcontrollers to automate data collection and expand the sample size through multiple data channels. Multiple data channels enable the user to not only increase their sample size, but also allows for multiple treatments to be run simultaneously in a single experiment. Furthermore, we created an R script to automatically visualize the data as a bubble plot, while also calculating the median day and time of emergence. The current system was designed using 3D printing so the user can modify the system to be adjusted for different species of insects. The goal of this protocol is to investigate important questions in chronobiology and stress physiology, using this precise and automated system to measure insect emergence patterns.

Measurement of insect emergence patterns requires precision. Existing systems are only semi-automated and sample size is limited. We addressed these issues by designing a system using microcontrollers to precisely measure the time of emergence of large numbers of emerging insects.

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of ...

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized into five different toxinotypes, A through E, based on the carriage of four major toxin genes. The prevalence and distribution of these various toxinotypes is understudied, especially their pervasiveness in American retail food. Of particular interest to us are the type B and D strains, which produce epsilon toxin, an extremely lethal toxin suggested to be the environmental trigger of multiple sclerosis in humans. To evaluate the presence of different C. perfringens toxinotypes in various food samples, we developed an easy method to selectively culture these bacteria without the use of an anaerobic container system only involving three culturing steps. Food is purchased from local grocery stores and transported to the laboratory under ambient conditions. Samples are minced and inoculated into modified rapid perfringens media (RPM) and incubated overnight at 37 &#176;C in a sealed, airtight conical tube. Overnight cultures are inoculated onto a bottom layer of solid Tryptose Sulfite Cycloserine (TSC) agar, and then overlaid with a top layer of molten TSC agar, creating a &#34;sandwiched&#34;, anaerobic environment. Agar plates are incubated overnight at 37 &#176;C and then evaluated for appearance of black, sulfite-reducing colonies. C. perfringens-suspected colonies are removed from the TSC agar using sterile eye droppers, and inoculated into RPM and sub-cultured overnight at 37 &#176;C in an airtight conical tube. DNA is extracted from the RPM subculture, and then analyzed for the presence of C. perfringens toxin genes via polymerase chain reaction (PCR). Depending on the type of food sampled, typically 15&#8211;20% of samples test positive for C. perfringens.

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

The objective of this protocol is to detect different Clostridium perfringens toxinotypes in locally purchased foods, particularly epsilon toxin producing strain types B and D, without the use of anaerobic chambers.

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized ...