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 growers within the mid-Atlantic United States, 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, emerging surveillance techniques utilizing a genetic resource known as environmental DNA (eDNA) have a proven track record in aquatic systems, which determines presence of target species at abundances far below what direct monitoring can accomplish. I have adapted these aquatic eDNA techniques for terrestrial uses to develop a spatially fine resolution detection protocol for agriculture pests, specifically BMSB. More specifically, I 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 I, in collaboration with others, developed and tested specifically for BMSB. Results utilizing this assay thus far indicate that BMSB indeed does leave genetic material on crop surfaces that are detectable in both field and laboratory trials. In addition, conducting surveillance in this way greatly outperformed traditional monitoring practices, indicating the ability to detect small populations more effectively than blacklight and pheromone traps. 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.     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.

a.     Can BMSB eDNA be detected on crop surfaces, or within suspected feeding sites on produce?

– Under this objective our goal was 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. I found that time was not a factor, since even a single defecation event by BMSB resulted in a positive detection and occurred at all tested time intervals. However, I found that the forensic grade swabs utilized were causing detection problems, and have yet to determine why exactly this is the case. Standard cotton swabs that were UV sterilized within the lab performed well, and provided adequate detection results, indicating the forensic grade swabs purchased must have had a manufacturing problem that was interfering with our results. When planning the field experiments, I realized that swabbing crop surfaces would have subjected detection probabilities to sampling effects, since not every crop surface tested would have had eDNA deposited on it. To combat this I would have had to test a very large number of crops, which would have resulted in a very large number of samples that require testing, inflating the cost of this method beyond what may have been practical.

b.     Can BMSB eDNA be found in the top soil directly beneath crops?

– In this objective our goal was to gather a small amount of topsoil from directly underneath crops. The assumption here was that during rain, irrigation, or just gravity that genetic material would fall off the plant and be left behind in the soil. While testing this concept within a controlled laboratory setting I found that BMSB eDNA from small exuvia fragments and a single defecation bouts were positively detected in as much as 10g of loamy soil. When testing this in the field I found that soil samples directly after a rain event were also positive, indicating the conceptual framework of eDNA deposition from crops to soil may indeed be correct. Further testing is being conducted to see how eDNA may move through the soil layers to determine if there is loss of material with continued water movement through the soil substrate.

c.     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. Laboratory experiments where crop samples that had BMSB defecate on their surface showed that the 10 micron filters captured more material containing target DNA than the 1 micron filter, likely due to rapid saturation of the smaller 1 micron filter. I found this result to be consistent when tested in our positive control field, which was also beneficial since a larger pore size would not rapidly saturate when free floating organic matter was present within the collected water samples. One limitation that was brought to my attention was that standard municipal water contains chlorine or chloramines, which rapidly break down the DNA and provide no signal both in the lab and in the field. Fortunately, I learned that my experiment sites did not use municipal water for their growing purposes, so this was not an immediate concern. When I tested this concept in the lot abundance site I found several positive detections over the course of two weeks. In addition, I was able to determine specifically which crop stands had positive detections for BMSB.

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

– In this section I 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). When I conducted this test in the New Jersey site I found detections with both the eDNA method and the traditional monitoring traps. This was likely due to the high abundance of BMSB already in the area, which was to be expected. However, when I conducted the same experiment in the low abundance location in New Hampshire I found several positive detections over the course of two weeks utilizing the eDNA method. Conversely, I did not see any positive detection for BMSB in the blacklight traps, and found only a single nymph in the pheromone traps on the last day of testing. This result indicates that the eDNA method indeed is more capable of detecting incipient BMSB populations than conventional methods.

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

– In light of the results I received in the previous objectives I have found that utilizing eDNA surveillance methods was superior to traditional practices when surveying for BMSB populations while abundance is still very low. Since the wash water results proved to be very effective and was the most cost efficient, I am moving forward in developing a surveillance protocol utilizing eDNA. This protocol will take into consideration the use of municipal water that contains chlorine and chloramines, possible ways to counteract this problem, and potential alternatives that would bypass the problem altogether.

Accomplishments/Milestones

• I found that the forensic grade cotton swabs were ineffective, possibly due to a manufacturing error. When standard cotton swabs that were US sterilized within the lab were used instead, I found positive results where expected, when there previously was none. However, when I began working with this swabbing concept it quickly became apparent that I was falling into a sampling problem when conducting surveillance in this manner. In order to adequately acquire a positive result at any site I would have to physically find a crop that has BMSB DNA on its surface and swab it, which is not time or cost effective. As a result, I concluded that this method would not be ideal for surveillance on crops. However, its utility can be useful elsewhere, such as testing equipment or other surfaces that ctops pass through regularly and in high concentration. More experiments on this front will be necessary, but the results acquired from swabs thus far show that it can be promising.
• The results from my was water tests showed that the larger filter pore size of 10 microns performed significantly better than the 1 micron pore size filters. This was somewhat unexpected at the time, until I realized that it might be due to rapid oversaturation of the 1 micron filter, where the 10 micron filter can hold more material due to its larger pores. Additionally, the 10 micron filter would likely perform significantly better in the field because the free-floating organic matter and soil particles would not clog the surface rapidly.
• Once I found an appropriate soil kit I began testing the concept of detecting BMSB DNA within the soil. Immediately I found that even a small fragment of BMSB, a small exuvia fragment, or a single defecation bout was easily detectable in as much as 5g of loamy soil. However, I soon realized that the BMSB DNA may not sit on the surface of the soil, but possibly may move through it as rain or irrigation of crops occurs. Therefore, experiments are still underway regarding how the DNA may move though the soil column during rain or irrigation before a protocol can be designed around it.
• Prior to carrying out my field experiments I learned about how municipal water was treated, the chemicals ever present within it, and its use on most commercial farms. This treatment of municipal water breaks down DNA rapidly, so I found it detrimental to my research. As a result, I immediately checked with my study sites to see if they utilize municipal water and to see in what ways adjustments could be made so I could continue with my work. I soon learned that these sites did not use municipal water, making them ideal for my research to continue uninhibited.
• When I began conducting field experiments in my high abundance site in New Jersey, I initially received no positive detections via my eDNA method, despite the nearby pheromone traps clearly showing they were present. This trend continued into my first week’s sample collection at my low abundance site in New Hampshire. When I returned for a second week I set up an experimental positive control where a single wash container was spiked with increasing BMSB DNA levels and tested. This allowed me to determine if there was some sort of inhibition taking place that was interfering with my eDNA technique, or if perhaps there were no BMSBs at the site to give a positive result. The container was kept isolated from the other washing containers to ensure no cross contamination would occur. After testing the newly collected samples and my experimental positive control I again found no positive detections, indicating that something was inhibiting my eDNA tool when in the field. After looking through the literature I found that organic matter inhibits qPCR unless neutralized with an environmental mastermix. After acquiring and retesting all samples with this new mix I found that all samples at my high abundance site were positive, all experimental positives at my low abundance site were positive, and several samples (representing different crop stands) across all days for each week were positive. In addition, since there was only a single positive detection with the pheromone traps on the last day of testing, and there were no positive detections with the blacklight trap, this indicated that my eDNA method was much more effective at detecting the low abundance population compared to the traditional traps.

Impacts and Contributions/Outcomes

Upon receiving the results from the low abundance site in New Hampshire, the farmers were immediately notified of the small BMSB population growing in their fields. As a result they have since begun discussions with local extension specialists in order to proactively manage BMSB populations before they become problematic. This proactive approach will allow them to prevent populations from booming and causing significant losses to their crops, to which are sold directly to the local community, resulting in minimal economic losses. Additionally, their proactive approach will allow them to utilize fewer pesticides than they would have had they reactively attempted to manage a larger growing population on their fields. Furthermore, due to the eDNA technique’s success in determining which crop stands contained BMSB DNA signatures, they could focus their efforts on the infected crop stands as well as those directly adjacent, rather than attempting to spray all their fields simultaneously.

Providing information to farmers regarding pest presence and precise locations of detection would not only relieve farmers from the financial burden of spraying all their fields but also result in reduced chemical input into the environment as well. The utility of eDNA based surveillance can prove to be a phenomenal addition to a farmer’s sustainability toolkit, because it would have minimal impacts on current farm practices, reduce monetary and environmental costs of pesticide use, and allow proactive decision making for managing dangerous crop pests on their plots.

Collaborators: