Harmonic Radar Tags for Insect Tracking: Lightweight, Low-cost, and Accessible

Riepilogo

This protocol presents the methods for creating, attaching, and tracking harmonic radar tags for insects.

Abstract

Using tracking devices to follow animal movements has long been a standard approach for studying the behavior and ecology of vertebrates. Until relatively recently, these tracking methods have been unavailable to entomologists due to the prohibitively large size and mass of the tracking devices that must be attached. Technologies applied to insect tracking include radio telemetry, Bluetooth, RFID, and harmonic radar. Among these, radio telemetry is the most used method to track larger insects. It offers a longer detection range and unique signals for each tracked insect but requires expensive tags that have limited shelf and field life and are heavy, generally at least 150 milligrams. By contrast, harmonic radar tags, first used in 1986, are lighter by at least an order of magnitude, inexpensive, and have detection ranges that allow field tracking of many smaller insects. Here, we demonstrate the use of an off-the-shelf transceiver marketed for backcountry rescue. Together, inexpensive tags and off-the-shelf transceivers make this insect-tracking technique highly accessible to entomologists studying insect behavior and ecology. In this article, we will touch on creating tags, attaching tags to insects, tracking these tagged insects, and present some representative tracking results.

Introduzione

Employing tracking devices to study the movements of vertebrates has long been a standard approach for collecting data related to the behavior and ecology of larger animals such as mammals, birds, and fish, with tracking primarily utilizing radio telemetry and GPS tracking¹. However, until relatively recently, these tracking methods have been unavailable to entomologists due to the prohibitively large size and mass of the tracking devices that must be attached². Technologies applied to insect tracking include radio telemetry, Bluetooth, RFID, and harmonic radar^2,3. Of these, radio telemetry is by far the most used method to track larger insects². It offers a longer detection range and unique signals for each tracked insect. This method, however, requires expensive tags that have limited shelf and field life and are heavy, generally at least 150 mg. Bluetooth is the least developed technique currently, with smaller tags than radio telemetry but a shorter range as well³. RFID tags can be very small in size and are quite inexpensive but have the shortest range (often only a few mm)⁴. Insect tagging with RFID tags has most commonly been used to study bee workers entering and leaving the hive⁵. By contrast, harmonic radar tags are lighter by at least an order of magnitude than radio telemetry tags, inexpensive, and have detection ranges that allow field tracking of much smaller insects⁶ (Figure 1).

Harmonic radar uses a transceiver that emits a signal that energizes the tag carried by the insect and receives the frequency-doubled return signal sent by the tag^7,8,9 (Figure 2). While a number of studies have described the use of harmonic radar tags with medium-sized insects^{6,10,11,12,13}, there is no proper demonstration of how to fabricate a tag. In this article, we provide step-by-step instructions on how to fabricate two different sizes of harmonic radar tags, each constructed from a Schottky diode and nitinol wire. Smaller tags are lighter and work better for smaller insects⁶, while the larger tags have a greater range and can be accommodated by larger insects¹¹. The nitinol wire is a critical design component because this wire is flexible, thereby letting the tagged insect move relatively freely. It also returns quickly to a straight orientation, allowing maximum signal return^6,10,11. We also demonstrate the use of an off-the-shelf transceiver (see 'Obtaining signals' section below) marketed for backcountry rescue^7,9,14. Together, inexpensive tags and off-the-shelf transceivers make this insect-tracking technique highly accessible to entomologists studying insect behavior and ecology. In this article, we touch on making tags, attaching tags to insects, tracking these tagged insects, and provide some representative tracking results.

Protocollo

1. Preparation

1. Assemble all the materials needed for tag making: nitinol wire (0.0254 mm for small tags, 0.0762 mm for large tags), Schottky diodes (one per tag), conductive adhesives (silver conductive epoxy is suggested for maximum strength, silver paint for lighter tags) and 3D printed jigs to be used for wire cutting and tag assembly (see Table of Materials).
2. Ensure the availability of two different-sized nitinol wires, 25 µm and 75 µm, to be used in small and large tags, respectively.

2. Large tag

NOTE: This section presents the assembly of large tags. It is important to have two wires of the correct antenna length (8.25 cm to match the transceiver frequency) for the large tags.

1. Use the measuring jig to reproducibly cut the wires to the desired length (8.25 cm) using a pair of scissors.
   1. First, use the single-sided tape to secure the wire to the jig.
   2. Wrap the wire around the jig (20-30 times max), maintaining light tension.
   3. Cut the wires in the channel using scissors. The size of the wire obtained is 8.25 cm.
   4. Then carefully remove the wires from the cylinder.
      NOTE: Two wires are needed for each tag.
2. Prepare the diode for wire attachment.
   1. Secure the diode packaging to the microscope using tape. Pull back the cover using forceps and remove the diodes with double-sided tape.
   2. Place double-sided tape over the packaging to remove diodes easily.
      NOTE: At this point, all the diodes are in the correct orientation, with contacts facing up to facilitate antenna bonding.
3. Affix the tape to the jig with the diodes facing up. Place cardstock parallel to the diodes on each side and smooth the edges to prevent the antenna from sticking to the tape.
4. Align the wires next to the diodes in preparation for the attachment and under a microscope, position the wires so that they are each touching a single electrical contact.
   NOTE: Wires should not touch both electrical contacts.
5. Apply conductive material to each contact using an insect pin. Ensure to cover both the contact point and the wire.
6. Take care not to spread the conductive material between each contact. If the conductive material blends together or touches the other wire or contact, this will greatly reduce the signal strength of the tag.
7. Reapply conductive material as needed to secure each wire and ensure electrical conductivity.
8. Allow tags to stay undisturbed for several hours to let the conductive material harden.

3. Small tag

NOTE: This section presents the assembly of small tags.

1. Cut wires to lengths of 4.1 cm.
2. Attach the wire to the jig with the one-sided tape, being careful not to over-tension the wire.
3. Wind the wire around the jig and cut it along the groove with scissors.
   NOTE: Each tag requires two segments of the wire prepared above.
4. Prepare the diodes for attachment. Place double-sided tape on the jig and transfer the diodes to the tape with forceps, maintaining an appropriate space between them.
5. Under a microscope, ensure contacts are facing up, facilitating the alignment of wire and diode. Realign these as needed, and ensure they are pressed into the tape to prevent movement during the assembly process.
6. Place single-sided tape along diodes to elevate the wire at the level of contact and prevent it from sticking to the underlying tape. Smooth down the tape and place the wires in the general vicinity of the diode.
7. Under the microscope, place the wire on each contact point with care to prevent wire overlap.
8. Apply the conductive material to the contact and wire and ensure that the conductive material does not flow together and flood the diode plane.
9. Allow tags to set undisturbed for several hours to let the conductive material harden.

4. Large tag attachment - Queensland longhorn beetle (QLB)

1. Wear appropriate PPE to protect from UV exposure (protective eyewear is recommended).
2. Secure the beetle to facilitate access to the attachment site. Hold the beetle gently, applying slight pressure to the table surface.
3. Apply a drop of UV adhesive to the attachment site.
4. Orient the tag and place the diode with the electrical contacts facing down onto the thorax.
5. Once satisfied with the position of the tag, cure the glue using UV light at a variety of different angles for a total of 5 to 10 s. This UV adhesive has been previously used to attach tags to larger insects without apparent negative effects of UV exposure¹¹.

5. Small tags attachment - tephritid fruit flies

1. Wear appropriate PPE to protect from UV exposure (protective eyewear is recommended).
2. Anesthetize flies at 4 ^oC. Typically, flies experience cessation of movement after 10-15 min.
3. Dispense a drop of glue and immerse the diode of the tag into the UV adhesive. Make sure to roll the diode in the glue to ensure full coverage. Use care to prevent excess adhesive from coating the wire.
4. Secure the insect between the thumb and forefinger to present the attachment site.
5. Orient the tag and place it longitudinally on the dorsal side of the fly thorax. Move the tag back and forth to spread the adhesive and ensure a secure connection. Use care not to apply glue to the head or wings.
6. When satisfied with the placement of the tag, cure the adhesive using a UV light. Several studies have used this UV adhesive to attach tags to flies without apparent negative effects of UV exposure^6,10,12,13.

6. Obtaining signals

1. Test the flight ability of insects after tag attachment. Do this by testing in a flight tube.
2. Then let the flies take off into their habitat.
3. Use a transceiver (see Table of Materials) to obtain signals from large and small insects.
   NOTE: When aligned with a tag, the transceiver produces a sound. When the transceiver is rotated 90 degrees, a weak or no signal is obtained due to the misalignment of the tag and the transceiver.

Risultati

Detection range and tag size are probably the two most important features of insect tracking tags. Here, we present representative results for the maximum detection range of the large and small tags fabricated in this article. Maximum detection ranges were determined by securing the tags longitudinally to a thick wooden dowel (~1.5 m) with double-sided tape. The maximum distance that tag signals could be detected was then determined by moving the transceiver away from the tag until no signal was audible. Ten tags of each size were tested in an open field. The maximum detection distance for the larger tags is roughly four times that of the smaller tags (Table 1). Misalignment of the tag and transceiver, along with vegetation interference, often reduces the detection range under field conditions. In our experience, we assume a detection distance of ~10 m for larger tags and ~5 m for small tags when working in tree field crops.

Finally, we would like to share the results from a representative tracking study with Queensland longhorn beetle (QLB). The aim of this study was to investigate the movement ecology of QLB, as little is known about where adult beetles spend time in the environment. As the beetles are cryptic, it is quite difficult to locate them in the environment without a tracking technique such as HR. This study was conducted in a stand of kukui trees (Aleurites moluccana) on the Big Island of Hawaii. Multiple beetles were tagged and released during the course of this study. Beetles were then located using the tracking techniques demonstrated earlier in this article. An example of the path taken by one beetle over the course of 10 days can be seen in Figure 4. Note that the beetle was tracked to multiple locations, including high in kukui trees and to dead leaves. This was an important finding of this study. As QLB can be highly cryptic, farmers have often asked about where the beetles were "hiding" around the trees. By tracking individual QLB, we were able to determine that beetles were often concealed in dry leaves, making them difficult to detect. This study offers important new insights into where QLB adults are spending time both during the day and movement during the night.

Figure 1: Comparison of devices commonly used to track insects. Please click here to view a larger version of this figure.

Figure 2: Harmonic radar works by sending a signal at one frequency (blue) and then receiving the returned (frequency doubled) signal (red). Please click here to view a larger version of this figure.

Figure 3: Signal strength is related to the orientation of the tag and transceiver. Please click here to view a larger version of this figure.

Figure 4: Representative results from tracking a single Queensland longhorn beetle in kukui trees. Please click here to view a larger version of this figure.

Large tags	Small tags
64 ± 1 m	12 ± 1 m

Table 1: Maximum detection distances (mean ± SE, m) for insect harmonic radar tags (N = 10).

Discussione

In this protocol, we describe and demonstrate how to fabricate two sizes of harmonic radar tags. In addition, we have shown how to attach these tags to insects using UV-cured adhesives and demonstrated transceiver signal strength. This protocol has been refined to allow for the streamlined fabrication of many low-cost HR tags with minimal specialized laboratory equipment. Additionally, the protocol should be accessible to researchers with experience manipulating objects under a dissecting microscope. We recommend that persons attempting to make tags begin with larger tags to develop skills in manipulating the diodes and applying the conductive adhesives. These skills can be difficult at first, but we have found from experience (teaching over 20 people) that the learning curve allows people to progress relatively quickly to making smaller tags with speed and reproducibility.

While the fabrication of two tag sizes was shown, we have also made tags with an intermediate-sized diode⁶, using both the smaller and larger diameter nitinol wire. Different applications, such as insect size and required detection range, influence the choice of diode and wire sizes.

The tag design used in this protocol is simpler than others used to track insects^15,16,17,18. A commonly used design is to have a loop in the antenna, thereby creating a DC short across the diode^18,19. Tags with loops in their antennas are usually mounted vertically on the thorax as opposed to longitudinally, as shown in this protocol. Vertical orientation potentially allows better alignment of tag and transceiver while the insect is in flight (as insects generally do not fly upside-down or with their legs toward the side). However, vertical tag orientation moves the center of the tag mass away from the insect's center of mass, thereby potentially having a greater influence on balance and flight ability.

Materiali

Name	Company	Catalog Number	Comments
Large assembly jig		https://www.thingiverse.com/thing:6784793
Large wire jig		https://www.thingiverse.com/thing:6784784
Nitinol Wire (Small)	Fort Wayne Metals (Fort Wayne, IN, USA)	17822174
RECCO R9 Handheld Detector	RECCO AB (Lidingo··, Sweden)		Frequencies of operation: Europe, 866.9 MHz (transmitting),1733.8 MHz (receiving); US, 902.85 MHz (transmitting), 1805.7 MHz (receiving)
RF Schottky Diode (Small)	MACOM Technology Solutions (Lowell, MA, USA)	MA4E2501L-1290	Puchased via Richardson RFPD
Schottky Diode (Large)	RECCO AB (Lidingo··, Sweden)
Silver Conductive Epoxy Adhesive	MG Chemicals	12642-14  (Amazon: 8331D)	Puchased via Amazon
Small assembly jig		https://www.thingiverse.com/thing:6784791
Small wire jig		https://www.thingiverse.com/thing:6784775
Super-Elastic Nitinol Wire (Large)	McMaster-Carr (Brookpark, Ohio, USA)	8320K31	0.003" Diameter x 30 Feet Long
Ted Pella Inc Silver Conductive Epoxy	Thermo Fisher Scientific (Waltham, MA, USA)	NC9959045
UV Adhesive	Bondic (Niagara Falls, NY, USA)		Purchased via Amazon and/or Bondic online (notaglue.com)