Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus (Murayama), Using a Low-Cost, Small, and Easily Constructed Flight Mill

Podsumowanie

We developed a low cost and small flight mill, constructed with commonly available items and easily used in experimentation. Using this apparatus, we measured the flight ability of an ambrosia beetle, Platypus quercivorus.

Streszczenie

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). Therefore, knowing the dispersal capacity may help inform trapping/tree removal efforts to prevent this disease more effectively. In this study, we measured the flight velocity and duration and estimated the flight distance of the beetle using a newly developed flight mill. The flight mill is low cost, small, and constructed using commonly available items. Both the flight mill arm and its vertical axis comprise a thin needle. A beetle specimen is glued to one tip of the arm using instant glue. The other tip is thick due to being covered with plastic, thus it facilitates the detection of rotations of the arm. The revolution of the arm is detected by a photo sensor mounted on an infrared LED, and is indicated by a change in the output voltage when the arm passed above the LED. The photo sensor is connected to a personal computer and the output voltage data are stored at a sampling rate of 1 kHz. By conducting experiments using this flight mill, we found that P. quercivorus can fly at least 27 km. Because our flight mill comprises cheap and small ordinary items, many flight mills can be prepared and used simultaneously in a small laboratory space. This enables experimenters to obtain a sufficient amount of data within a short period.

Wprowadzenie

Animals migrate long distances in search of food and mates. Migrating animals might sometimes carry undesirable companions. The female ambrosia beetle, Platypus quercivorus (Murayama), is a known vector of the fungal pathogen, Raffaelea quercivora Kubono et Shin-Ito. This pathogen causes mass mortality of Fagaceae trees (Japanese oak wilt) and a high level of mortality¹. Since 1980, this disease has been expanding throughout Japan, and has become a serious problem².

P. quercivorus is a small insect (4-5 mm in body length and 4-6 mg in body weight), and yearly expansion of the disease suggests that they are capable of flying up to several km^3,4. The male P. quercivorus locates a host tree and releases an aggregation pheromone that attracts both males and females⁵. Consequently, the host tree is mass attacked by conspecifics, and eventually dies. The male bores a tunnel inside the tree after landing and a pheromone-attracted female enters the tunnel and lays eggs. The hatched P. quercivours grow in the tunnel until they become adults. Adults emerge and disperse to locate new hosts. Thus, expansion of the disease is possibly related to the migratory ability of this beetle. However, the extent to which the beetle can fly is still unclear. In addition, females are bigger than males⁶ (female: 4.6 mm, and male: 4.5 mm) and male beetles search for a target tree, enter the tunnel inside the tree, and then attract the female. Considering these sexual differences in the body size and role of flight in their life, sexual differences may exist in flight ability, but the difference in ability remains unclear.

In general, it is extremely difficult to measure migratory ability in the field, especially flight ability, due to the wide range of the migratory area. Migratory ability has been measured in laboratories under tethered conditions, such as a flight mill system, for over 60 years^{7,8,9,10,11,12,13}. Flight mill systems have shown that some insects have the ability for long distance flight. For example, the longest flight distance of the mountain pine beetle in a flight mill was over 24 km¹⁴, and Tetrastichus planipennisi Yang flew maximally over 7 km¹⁵. Although the flight mill is a commonly available tool, biological assays with a living animal often result in considerably large individual differences. To overcome this, many measurements, repeated multiple times, are required to obtain reliable estimates of mean dispersal capacity. Therefore, multiple individuals should be used at the same time for the quick collection of a sufficient amount of data. However, simultaneous experiments require a larger space, multiple experimental setups, and are costlier when compared to a single measuring system. Hence, the flight mill must be low cost, should be easily built with commonly available items, and compact in size. Furthermore, the experimental procedure should not be complicated or need a skillful operator.

In this study, we assembled a small, low-cost flight mill (Figure 1 and Figure 2) that could be easily used in experimentation, and measured the flight ability of the ambrosia beetle, P. quercivorus.

Protokół

1. Construction of a Flight Mill

1. Construction of a flight mill apparatus
   1. Cut off the plastic part from a needle (metal part: 40 mm in length and 0.25 mm in diameter; plastic part: 22 mm in length and 2 mm in diameter) with nippers (Figure 3).
   2. Fix this needle with an untreated needle in the shape of a cross with epoxy resin adhesive (Figure 3), referring them as a flight mill arm and an axial needle.
      NOTE: For an axial needle, the untreated side should be a bottom side. The uncovered tip of the flight mill arm is for gluing a beetle (Figure 1B and Figure 3).
2. Construction of the base
   1. Make a small dimple on the surface of a thin stainless metal plate (5 cm x 5 cm) by hammering a nail to prevent the axial needle from sliding horizontally (Figure 4).
      NOTE: The actual dimensions of the metal plate are not critical, and another material is possible, but avoid using any soft material; otherwise, the needle will be stuck, preventing the mill from revolving.
   2. Place and fix the metal plate on the wooden board (wooden base) with adhesive tape.
   3. Bend a steel plate to make it double L-shaped (Figure 1C and Figure 2A).
      NOTE: It was convenient to use an L-shaped metal plate for fixing furniture on the wall. Another convenient point in support of using this kind of plate was that the plate already had many holes. Holes were used for screwing, and also fixing a snap button (Figure 1A and Figure 4).
   4. Make a cylinder by cutting the tip of a disposable plastic pipette (height = 1 cm, outside diameter (o.d.) = 4 mm, interior diameter (i.d.) = 2 mm) for guiding an axial needle (Figure 2A and Figure 4).
   5. Put and fix the double L-shaped plate and the cylinder on the metal plate (Figure 2A and Figure 4).
3. Construction of the sensing apparatus
   1. Bend a metal plate to make it L-shaped to make a top plate.
      NOTE: It was convenient to use an L-shaped metal plate for fixing furniture on the wall (Figure 5B-C). If so, you can skip this step.
   2. Put a small metal cap (5 mm in length and 1 mm in diameter) on the top plate (Figure 2D-E, Figure 4, and Figure 5A).
      NOTE: As a cap, we used a snap button. It passed through a hole in the L-shaped plate (Figure 4).
   3. Fix a photo sensor on the L-shaped plate (Figure 4 and Figure 5A). Screwed a circuit substrate for the sensor on the L-shaped plate to save space (Figure 2D-E, and Figure 4).
   4. Glue an infrared LED (150 mW) on a small magnet together with a circuit substrate for the LED (Figure 1A and Figure 2A).
   5. Place the LED (150 mW) on the base plate beneath the photo sensor (Figure 1A and Figure 2A).
4. Construction of the holder
   1. Bend a metal plate to make it L-shaped.
      NOTE: It was convenient to use an L-shape metal plate for fixing furniture on the wall (Figure 5B-C). If so, you can skip this step.
   2. Fix the plate on a wooden board (wooden wall) with screws (Figure 1C, Figure 4, and Figure 5B). The height of the wooden board is not critical, it was 7 cm in this study.
5. Connecting cables
   1. Connect the photo sensor to an analog input channel (AIN) of an A/D converter via normal electric cables.
      NOTE: It is helpful if all the cables are bundled and fixed on the L-shaped plate (Figure 5B-D) because a messy workspace often prevents fine manipulation throughout the experiment.
   2. Connect the A/D converter to a personal computer (PC) via a USB cable.

2. Experimental Procedure

1. Collect all freshly emerged P. quercivorus adults from a dead Quercus crispula Blume (Fagales: Fagaceae) tree in the morning (7-9 am) of the day on which the experiment is to be performed.
   NOTE: Do not use beetles collected in the previous day. More than 100 beetles came out every day and newly emerged beetles were checked daily. See a reference¹⁶ for detailed methods on collecting beetles.
2. Put a beetle on ice for anesthetization. Avoid getting the beetle wet; otherwise, it will be difficult to complete the following procedure. Perform all subsequent procedures on ice.
3. Place a small amount of one component of the instant glue (jellylike glue) on the beetle's pronotum with the mill arm, and keep the mill arm in contact with the pronotum.
   NOTE: The jellylike glue will dry slowly if this glue is used alone. However, this glue functions quickly when two components are mixed (Table of Materials). The other component (liquid glue) will be used in the next step.
4. Add a small amount of the other component of the glue (liquid glue) using a fine needle or stick. Ensure that the wings are free from glue (Figure 1B). The liquid glue is used to facilitate the hardening of the jellylike glue.
5. Adjust the cross-shaped needle into the flight mill (Figure 6) by using a magnet to hold the L-shaped plate (top plate) on the other L-shaped plate. Simply slide the top plate when adjusting the height of the top plate for the needle. Insert the upper tip of the axial needle into the hole of the snap button on the top plate (Figure 5A), and place the other tip into the guide on the base plate (Figure 6).
6. Adjust the position of an IR LED beneath the sensor.

3. Obtain and Analyze Data

1. Record the amplified output signal from the photo sensor and store it in the PC through the A/D converter by using commercial software with a sampling rate of 1,000 points/s (Figure 7A) (for A/D converter and software, Table of Materials).
2. Start the software DAQFActoryExpress.
3. Click a cross (+) mark on the LOGGING icon in the Workspace window.
4. Right click the logging set name and select Begin Logging Set.
   NOTE: The software continues logging and saving data.
5. To stop recording, right click the logging set name and select End Logging Set to save a .csv file.
6. Extract the passing time of the flight mill arm above the IR LED using an appropriate software by detecting times only when the recorded voltage exceeded the threshold (0.5 V).
   NOTE: Because some software (e.g., MS Excel) can read a created .csv file, use a familiar software depending on the study's purpose. If necessary, download the custom-made programs available via Github, https://github.com/HidetoshiIkeno/FlightMill. For further information about our programs as well as instructions to use the program, see the README file which is accompanied with the main program.

Wyniki

In these experiments, about 50% of the beetles applied to the flight mill showed one or more revolutions. When the plastic part passed a virtual line between the sensor and the LED, the recorded voltage changed from about 0 V to about 6.5 V, and the duration of a passing was within 10-20 ms, depending on the flight velocity. Therefore, a spike-like voltage change is observed as one revolution (Figure 7B). We defined flight as when the flight mill arm revolved...

Dyskusje

We developed a low-cost, easy-to-build, and compact flight mill for small insects such as P. quercivorus (4-5 mm in body length and 4-6 mg in body weight). Our flight mill comprised only ordinary items such as a needle, an IR LED, a photo sensor, instantaneous glue, etc., and did not require any sophisticated, expensive, or rare items such as computer-controlled electric devices. This enabled the easy and quick collection of necessary items and reduced experimental costs. Indeed, it cost only 1,000 JPY ...

Materiały

Name	Company	Catalog Number	Comments
needle	Seirin	J type No. 5 x 40 mm
epoxy resin adhesive	Konishi	#16113
metal plate			from a home improvement store
disposable plastic pipette			from a home improvement store
snap button			from a craft store
IR sensor	Hamamatsu Photonics	S7136
IR LED	OptoSupply	OSIR5113A	150 mW
custom-made program			downloadable from Github. URL: https://github.com/HidetoshiIkeno/FlightMill
instant glue	Toagosei	31204
A/D converter	LabJack Co.	U3-HV
DAQ software	AzeoTech	DAQFactoryExpress	download from AzeoTech Web page.