Development of a high-resolution surveillance protocol using eDNA for detection of brown marmorated stink bugs

Development of a high-resolution surveillance protocol using eDNA for detection of brown marmorated stink bugs

Summary

It is well established that early detection is the single-most effective strategy for control of invasive species. The brown marmorated stinkbug (BMSB, Halyomorpha halys), which has become a devastating pest to many northeastern farmers, was first detected in the US in 1996 and is currently still expanding across the continent. Current efforts at monitoring for the presence of BMSB on farms rely on capturing individuals using black light and pheromone traps, which may be producing false negatives while populations are at low abundance, thus delaying management actions that could otherwise effectively control populations on agricultural fields. However, an emerging surveillance tool known as environmental DNA (eDNA) has a proven track record in aquatic systems, which determines presence of target species at abundances far below what direct monitoring can accomplish. We have adapted these aquatic eDNA techniques for terrestrial uses to develop a fine resolution detection protocol for agriculture pests, specifically BMSB. More specifically, we will be testing individual crop surfaces, topsoil beneath crops, and crop wash water to survey for residual DNA deposition from BMSB to determine detection. This will primarily be accomplished by utilizing a genetic assay we developed and tested specifically for BMSB. Early results utilizing this assay indicate that BMSB indeed does leave behind genetic material that is detectable, however more work is required to further optimize the process for field application. Ultimately the use of eDNA will allow rapid-onset control to be implemented, which will allow small produce farms to maintain their profitability by keeping costs low, due to reduced pesticide use, while experiencing fewer crop losses.

Objectives/Performance Targets

1. 1. Determine if BMSB eDNA can be detected on small produce farms.

• • The goal for this part of the project is to determine the most effective sampling strategies that will allow us to detect BMSB eDNA on farms.

1. 1. Can BMSB eDNA be detected on crop surfaces, or within suspected feeding sites on produce? – Under this objective our goal is to utilize forensic grade swabs (i.e. sterile and DNA free) to collect DNA that may have been deposited by BMSB while on the surface of crops. Testing of crop surfaces began in a controlled laboratory setting by placing BMSB on individual fruits within a container for several predetermined time intervals and then swabbing the entire surface after. This was to determine not only if BMSB can be detected in such a way, but to identify what may be the minimum time required for a single BMSB to leave sufficient amounts of genetic material behind to be detectable. To date there have been no successful detections with this method, and reasons as to why (e.g. defective swabs) are being explored.

1. 1. Can BMSB eDNA be found in the top soil directly beneath crops? – In this objective our goal is to gather a small amount of topsoil from directly underneath crops. The assumption here is that during rain, irrigation, or just gravity that genetic material will fall off the plant and be left behind in the soil. Work on this front has been delayed as newly found literature has brought the use of the initially proposed extraction kit into question, thus current efforts to identify a successful and cost-effective replacement kit are underway. At current there are two products that hold promise, and they are being evaluated for not only cost but quantity and throughput of samples as well.

1. 1. Can BMSB eDNA be found within the water of crop wash stations? – In this objective the success of eDNA surveillance in aquatic systems was adopted and adjusted for use in a terrestrial setting in hopes of equivalent success. Further reading of the eDNA literature involving surveillance in aquatic systems has shown that the previously proposed filter pore size of 20-microns would trap an abundance of free-floating DNA, which is fine for community identification but provides problems when surveying for a specific target. This is because macro-organism targets shed and excrete biological material that contains DNA, meaning surveillance is being done on intact cells that fall into the 1-10 micron range. In light of this I set out to test both pore sizes. The experiment was conducted in a similar fashion to that of the swab experiment above (section 1a.), except rather than swabbing the surface the fruits were placed in buckets filled with a liter of water to rinse off any material on the surface. After being rinsed the fruits were removed from the water and discarded. These water samples were then run through the sample filtering system with the 1-micron pore size, which resulted in a positive detection of BMSB DNA. Testing and comparing detection between the 1-micron and 10-micron filters is being set up in a more laboratory controlled manner in order to better control for differences in the deposition of BMSB genetic material between individuals.

2. 2. Use eDNA to detect BMSB when they are so rare that direct monitoring fails to detect their presence.

• • In this section we will be comparing the effectiveness of the eDNA surveillance being developed here, and traditional monitoring tools currently being employed against BMSB (e.g. blacklight traps and pheromone traps). Work under this objective is scheduled for the field season in July and August of 2016 in two farms, each in a different state representing different BMSB abundance levels (New Jersey-high and New Hampshire-low/none). Until then laboratory work will continue to be carried out in order to employ the most optimized sampling protocols possible in the field. 

3. 3. Document and disseminate an eDNA surveillance protocol for detection of BMSB.

• • Once results have been acquired in the previous objective (2) I will compile all detection information and evaluate which sampling method(s) yielded the most cost effective approach for determining BMSB presence. If appropriate, we will then develop an eDNA surveillance protocol outlining best practices for farm-wide and crop-specific monitoring for BMSB presence.

Accomplishments/Milestones

Upon receiving the forensic grade cotton swabs we carried out the experiment for objective 1a, with an additional set of replicates to be used for objective 1c as well. After swabbing the fruits used in the experiment they were extracted and stored for further testing. The replicate experiment for water testing was frozen and stored due to a delay in acquiring all the necessary water-sampling components. Results from the cotton swabs were all negative, which seemed unusual in light of the early results obtained from the water test. One hypothesis is that the swabs may contain residual bleach from the manufacturing process, thus making them defective. Further testing of this is being carried out in order to confirm this, which will then result in the return of the swabs for a new product to test. The results of the water test were promising, and further laboratory work is being done in order to determine which filter pore size (1-micron or 10-microns) will provide the best detection and use for our application.

Work on detection of BMSB DNA in soil remains to be completed, however it was best that this particular objective not be rushed, do the inherent difficulty of carrying out genetic work with soil as pointed out in the literature. Experimental design of laboratory experiments for this objective have been outlined, and the two kits being considered will be ordered in early January in order to acquire early results on their performance and the performance of this sampling method.

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Impacts and Contributions/Outcomes

Though the project is still in an early phase, recent results from the aquatic sampling tests have shown remarkable sensitivity to BMSB DNA from just a single individual on a single fruit, in as much as a liter of water. This can considerably alter how growers look at water being used to wash crops. For instance, rather than discarding it or letting it flow away, it can be collected for testing at a local extension to determine if their farms have been invaded by BMSB. These early results have also enlightened me to explore and further understand how active BMSB is while around a crop (i.e. on said crop and/or feeding on it) for a fixed amount of time, and how this can influence its detection.

Collaborators: