We thank Mr. S. Fukaya, Mr. N. Okuda, and Mr. T. Ishino for helping with the experiments. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for Promotion of Science (No. 15K14755).

1. Construction of a Flight Mill
<ol>
	<li>Construction of a flight mill apparatus
	<ol>
		<li>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).</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Construction of the base
	<ol>
		<li>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.</li>
		<li>Place and fix the metal plate on the wooden board (wooden base) with adhesive tape.</li>
		<li>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).</li>
		<li>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).</li>
		<li>Put and fix the double L-shaped plate and the cylinder on the metal plate (Figure 2A and Figure 4).</li>
	</ol>
	</li>
	<li>Construction of the sensing apparatus
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>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).</li>
		<li>Glue an infrared LED (150 mW) on a small magnet together with a circuit substrate for the LED (Figure 1A and Figure 2A).</li>
		<li>Place the LED (150 mW) on the base plate beneath the photo sensor (Figure 1A and Figure 2A).</li>
	</ol>
	</li>
	<li>Construction of the holder
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Connecting cables
	<ol>
		<li>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.</li>
		<li>Connect the A/D converter to a personal computer (PC) via a USB cable.</li>
	</ol>
	</li>
</ol>
2. Experimental Procedure
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Place a small amount of one component of the instant glue (jellylike glue) on the beetle&#39;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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Adjust the position of an IR LED beneath the sensor.</li>
</ol>
3. Obtain and Analyze Data
<ol>
	<li>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).</li>
	<li>Start the software DAQFActoryExpress.</li>
	<li>Click a cross (+) mark on the LOGGING icon in the Workspace window.</li>
	<li>Right click the logging set name and select Begin Logging Set. 
	NOTE: The software continues logging and saving data.</li>
	<li>To stop recording, right click the logging set name and select End Logging Set to save a .csv file.</li>
	<li>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&#39;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.</li>
</ol>

<ol>
	<li>Kubono, T., Ito, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raffaelea+quercivora+sp.+nov.+associated+with+mass+mortality+of+Japanese+oak,+and+the+ambrosia+beetle+(Platypus+quercivorus).">Raffaelea quercivora sp. nov. associated with mass mortality of Japanese oak, and the ambrosia beetle (Platypus quercivorus).</a> Mycoscience. 43, 255-260 (2002).</li><li>Kobayashi, M., Ueda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wilt+disease+of+Fagaceae+trees+caused+by+Platypus+quercivorus+(Murayama)+(Coleoptera:+Platypodidae)+and+the+associated+fungus:+Aim+is+to+clarify+the+damage+factors.">Wilt disease of Fagaceae trees caused by Platypus quercivorus (Murayama) (Coleoptera: Platypodidae) and the associated fungus: Aim is to clarify the damage factors.</a> J Jpn For Soc. 87, 435-450 (2005).</li><li>Nunokawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+distribution+and+spreading+process+of+damages+caused+by+Japanese+oak+wilt+in+Niigata+Prefecture,+Japan+(in+Japanese).">Local distribution and spreading process of damages caused by Japanese oak wilt in Niigata Prefecture, Japan (in Japanese).</a> Bulletin of Niigata Prefectural Forest Research Institute. 48, 21-32 (2007).</li><li>Ohashi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+spreading+of+damages+caused+by+Japanese+oak+wilt+in+Gifu+Prefecture,+Japan+(in+Japanese).">Distribution and spreading of damages caused by Japanese oak wilt in Gifu Prefecture, Japan (in Japanese).</a> Bulletin of the Gifu Prefectural Research Institute for Forests. 37, 23-28 (2008).</li><li>Tokoro, M., Kobayashi, M., Saito, S., Knuura, H., Nakashima, T., Shoda-Kgaya, E., Kashiwagi, T., Tebayashi, S., Kim, C., Mori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+aggregation+pheromone,+(1S,4R)-p-menth-2-en-1-ol,+of+the+ambrosia+beetle,+Platypus+quercivorus+(Coleoptera:+Phatypodidae).">Novel aggregation pheromone, (1S,4R)-p-menth-2-en-1-ol, of the ambrosia beetle, Platypus quercivorus (Coleoptera: Phatypodidae).</a> Bulletin of FFPRI. , 49-57 (2007).</li><li>Nobuchi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platypus+quercivorus+Murayama+(Coleoptera,+Platypodidae)+attacks+to+living+oak+trees+in+Japan,+and+information+of+Platypodidae+(I).">Platypus quercivorus Murayama (Coleoptera, Platypodidae) attacks to living oak trees in Japan, and information of Platypodidae (I).</a> Forest Pest. 42, 2-6 (1993).</li><li>Clements, A. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+age,+sex,+and+reproductive+status+on+flight+by+the+tarnished+plant+bug+(Heteroptera:+Miridae).">Effects of age, sex, and reproductive status on flight by the tarnished plant bug (Heteroptera: Miridae).</a> Environ Entomol. 23, 80-84 (1994).</li><li>Sarvary, M. A., Bloem, K. A., Bloem, S., Carpenter, J. E., Hight, S. D., Dorn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diel+flight+pattern+and+flight+performance+of+Cactoblastis+castorum+(Lepidoptera:+Pyralidae)+Measured+on+a+flight+mill:+influence+of+age,+gender,+mating+status,+and+body+size.">Diel flight pattern and flight performance of Cactoblastis castorum (Lepidoptera: Pyralidae) Measured on a flight mill: influence of age, gender, mating status, and body size.</a> J Econ Entomol. 101 (2), 314-324 (2008).</li><li>Zhang, Y., Wyckhuys, K. A. G., Asplen, M. K., Heinpel, G. E., Wu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Binodoxys+Communis+parasitism+on+flight+behavior+of+the+soybean+aphid,+Aphis+glycines.">Effect of Binodoxys Communis parasitism on flight behavior of the soybean aphid, Aphis glycines.</a> Biol Control. 62, 10-15 (2012).</li><li>Sappington, T. W., Burks, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+flight+behavior+and+capacity+of+unmated+navel+orangeworm+(Lepidoptera:+Pyralidae)+Adults+related+to+age,+gender,+and+wing+size.">Patterns of flight behavior and capacity of unmated navel orangeworm (Lepidoptera: Pyralidae) Adults related to age, gender, and wing size.</a> Environ Entomol. 43, 696-705 (2014).</li><li>Attisano, A., Murphy, J. T., Vickers, A., Moore, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+flight+mill+for+the+study+of+tethered+flight+in+insects.">A simple flight mill for the study of tethered flight in insects.</a> J. Vis. Exp. (106), e53377 (2015).</li><li>Evenden, M., Whitehouse, L., C, M., Sykes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+flight+capacity+of+the+mountain+pine+beetle+(Coleoptera:+Curculionidae:+Scolytinae).">Factors influencing flight capacity of the mountain pine beetle (Coleoptera: Curculionidae: Scolytinae).</a> EnvironEntomol. 43, 187-196 (2014).</li><li>Fahrner, S. J., Lelito, J. P., Blaedow, K., Heimpel, G. E., Aukema, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+flight+capacity+of+Tetrastichus+planipennisi+(Hymenoptera:+Eulophidae),+a+classical+biological+control+agent+of+Agrilus+Planipennis+(Coleoptera:+Buprestidae).">Factors affecting the flight capacity of Tetrastichus planipennisi (Hymenoptera: Eulophidae), a classical biological control agent of Agrilus Planipennis (Coleoptera: Buprestidae).</a> Environ Entomol. 43, 1603-1612 (2014).</li><li>Pham, D. L., Ito, Y., Okada, R., Ikeno, H., Isagi, Y., Yamasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototactic+behavior+of+the+ambrosia+beetle+Phatypusquercirorus+(Murayama)+(Coleoptera:+Platypodidae)+before+and+after+flight.">Phototactic behavior of the ambrosia beetle Phatypusquercirorus (Murayama) (Coleoptera: Platypodidae) before and after flight.</a> J Insect Behav. 30, 318-330 (2017).</li><li>Wanner, H., Gu, H., Dorn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+value+of+floral+nectar+sources+for+flight+in+the+parasitoid+wasp,+Cotesia+glomerata.">Nutritional value of floral nectar sources for flight in the parasitoid wasp, Cotesia glomerata.</a> Physiol Entomol. 31, 127-133 (2006).</li><li>Rowley, W. A., Graham, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+age+on+the+flight+performance+of+female+Aedes+aegypti+mosquitoes.">The effect of age on the flight performance of female Aedes aegypti mosquitoes.</a> J Insect Physiol. 14, 719-728 (1968).</li></ol>

The authors have nothing to disclose.

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 ...

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 km3,4. 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 years7,8,9,10,11,12,13. 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.

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

measuring the flight ability of the ambrosia beetle, platypus quercivorus (murayama), using a low-cost, small, and easily constructed flight mill

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.

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...

Watch this Scientific Journal Video about Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus Murayama, Using a Low-Cost, Small, and Easily Constructed Flight Mill at JoVE.com

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

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.

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

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). ...

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7.6K Views.  University of Hyogo. Wij ontwikkeld van een low-cost en kleine vlucht molen, gebouwd met algemeen beschikbare items en gemakkelijk gebruikt in experimenten. Met behulp van dit apparaat, we het vermogen van de vlucht van een ambrosia kever, Platypus quercivorusgemeten.

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

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana1. Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of a variety of lignocellulosic feedstocks. Here we demonstrate the use of these low-cost protic ILs in the deconstruction of lignocellulosic biomass (Ionosolv pretreatment), yielding cellulose and a purified lignin. In the most generic process, the protic ionic liquid is synthesized by accurate combination of aqueous acid and amine base. The water content is adjusted subsequently. For the delignification, the biomass is placed into a vessel with IL solution at elevated temperatures to dissolve the lignin and hemicellulose, leaving a cellulose-rich pulp ready for saccharification (hydrolysis to fermentable sugars). The lignin is later precipitated from the IL by the addition of water and recovered as a solid. The removal of the added water regenerates the ionic liquid, which can be reused multiple times. This protocol is useful to investigate the significant potential of protic ILs for use in commercial biomass pretreatment/lignin fractionation for producing biofuels or renewable chemicals and materials.

The pretreatment of lignocellulosic biomass with protic low-cost ionic liquids is shown, resulting in a delignified cellulose-rich pulp and a purified lignin. The pulp gives rise to high glucose yields after enzymatic saccharification.

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of ...

Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte)

The western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is an economically important pest of corn in the northern United States. Some populations have developed resistance to management strategies including transgenic corn that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). Knowledge of western corn rootworm dispersal is of critical importance for models of resistance evolution, spread, and mitigation. Flight behavior of an insect, especially over a long distance, is inherently difficult to observe and characterize. Flight mills provide a means to directly test developmental and physiological impacts and consequences of flight in the laboratory that cannot be obtained in field studies. In this study, flight mills were used to measure the timing of flight activity, total number of flights, and the distance, duration, and speed of flights taken by female rootworms during a 22-h test period. Sixteen flight mills were housed in an environmental chamber with programmable lighting, temperature, and humidity control. The flight mill described is of a typical design, where a flight arm is free to rotate about a central pivot. Rotation is caused by flight of an insect tethered to one end of the flight arm, and each rotation is recorded by a sensor with a time-stamp. Raw data are compiled by software, which are subsequently processed to provide summary statistics for flight parameters of interest. The most difficult task for any flight mill study is attachment of the tether to the insect with an adhesive, and the method used must be tailored to each species. The attachment must be strong enough to hold the insect in a rigid orientation and to prevent detachment during movement, while not interfering with natural wing motion during flight. The attachment process requires dexterity, finesse, and speed, making video footage of the process for rootworms of value.

Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

Flight mills are important tools for comparing how age, sex, mating status, temperature, or various other factors may influence an insect&#8217;s flight behavior. Here we describe protocols to tether and measure the flight propensity and performance of western corn rootworm under different treatments.

The western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is an economically important pest of corn in the northern ...

Behavior

Building an Enhanced Flight Mill for the Study of Tethered Insect Flight

Makerspaces have a high potential of enabling researchers to develop new techniques and to work with novel species in ecological research. This protocol demonstrates how to take advantage of the technology found in makerspaces in order to build a more versatile flight mill for a relatively low cost. Given that this study extracted its&#160;prototype from flight mills built in the last decade, this protocol focuses more on outlining divergences made from the simple, modern flight mill. Previous studies have already shown how advantageous flight mills are to measuring flight parameters such as speed, distance, or periodicity. Such mills have allowed researchers to associate these parameters with morphological, physiological, or genetic factors. In addition to these advantages, this study discusses the benefits of using the technology in makerspaces, like 3D printers and laser cutters, in order to build a more flexible, sturdy, and collapsible flight mill design. Most notably, the 3D printed components of this design allow the user to test insects of various sizes by making the heights of the mill arm and infrared (IR) sensors adjustable. The 3D prints also enable the user to easily disassemble the machine for quick storage or transportation to the field. Moreover, this study makes greater use of magnets and magnetic paint to tether insects with minimal stress. Lastly, this protocol details a versatile analysis of flight data through computer scripts that efficiently separate and analyze differentiable flight trials within a single recording. Although more labor-intensive, applying the tools available in makerspaces and on online 3D modeling programs facilitates multidisciplinary and process-orientated practices and helps researchers avoid costly, premade products with narrowly adjustable dimensions. By taking advantage of the flexibility and reproducibility of technology in makerspaces, this protocol promotes creative flight mill design and inspires open science.

This protocol uses three-dimensional (3D) printers and laser cutters found in makerspaces in order to create a more flexible flight mill design. By using this technology, researchers can reduce costs, enhance design flexibility, and generate reproducible work when constructing their flight mills for tethered insect flight studies.

Makerspaces have a high potential of enabling researchers to develop new techniques and to work with novel species in ecological research. This protocol ...

Engineering

A Simple Flight Mill for the Study of Tethered Flight in Insects

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have shown that flight mills are valuable tools for the experimental study of insect flight behavior, allowing researchers to examine how factors such as age, host plants, or population source can influence an insects' propensity to disperse. Flight mills allow researchers to measure components of flight such as speed and distance flown. Lack of detailed information about how to build such a device can make their construction appear to be prohibitively complex. We present a simple and relatively inexpensive flight mill for the study of tethered flight in insects. Experimental insects can be tethered with non-toxic adhesives and revolve around an axis by means of a very low friction magnetic bearing. The mill is designed for the study of flight in controlled conditions as it can be used inside an incubator or environmental chamber. The strongest points are the very simple electronic circuitry, the design that allows sixteen insects to fly simultaneously allowing the collection and analysis of a large number of samples in a short time and the potential to use the device in a very limited workspace. This design is extremely flexible, and we have adjusted the mill to accommodate different species of insects of various sizes.

Flight in insects is influenced by a number of factors and the propensity to disperse is an important variable in understanding insect ecology and biological control strategies. We describe the construction and use of a simple, relatively inexpensive, and flexible flight mill for measuring parameters of tethered flight in insects.

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have ...

Neuroscience

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight insect behavior. This technology enables the development of an insect-machine hybrid system using a living insect platform described in this protocol. Moreover, this protocol presents the system configuration and free flight experimental procedures for evaluating the function of the flight muscles in an untethered insect. For demonstration, we targeted the third axillary sclerite (3Ax) muscle to control and achieve left or right turning of a flying beetle. A thin silver wire electrode was implanted on the 3Ax muscle on each side of the beetle. These were connected to the outputs of a wireless backpack (i.e., a neuromuscular electrical stimulator) mounted on the pronotum of the beetle. The muscle was stimulated in free flight by alternating the stimulation side (left or right) or varying the stimulation frequency. The beetle turned to the ipsilateral side when the muscle was stimulated and exhibited a graded response to an increasing frequency. The implantation process and volume calibration of the 3 dimensional motion capture camera system need to be carried out with care to avoid damaging the muscle and losing track of the marker, respectively. This method is highly beneficial to study insect flight, as it helps to reveal the functions of the flight muscle of interest in free flight.

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

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

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