Name: measuring the flight ability of the ambrosia beetle, platypus quercivorus (murayama), using a low-cost, small, and easily constructed flight mill
Uploaded: 2018-08-06T13:00:00
Description: 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

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. 
		NOT...

<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>

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. 
			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). ...

This method can help answer key questions in the entomology and forensic fields such as control of the migration of pests. The main advantage of this technique is to easily measure migratory distance and speed of a small insect in a laboratory under a controlled condition. Begin the construction of the spinning portion of the flight mill.First, remove the plastic portion of an acupuncture needle. A 40 millimeter long metal needle with a 0.25 millimeter diameter. Then, using an epoxy adhesive, make a cross with the modified needle and an unmodified needle of the same size.This makes the flight mill arm and an axial needle. Next, prepare a small metal plate. Hammer a dimple into the center of the plate where the axial needle will attach.Then, secure the plate to a wooden base using adhesive tape. Next, make a plastic cylinder to hold the spinning axis of the mill. Cut a plastic pipette to make it the same length with the double-L plate, which has an outside diameter of 4 millimeter and an interior diameter of 3 millimeter.Then attach these parts to the base plate with the cylinder over the dimple and with the double-L plate stabilizing the cylinder. Now make a holder from an L-shaped piece of metal and screw the metal to a vertical wooden board to stabilize the assembly. Into the hole of another L plate, fit a small metal cap that is about 10 millimeters long and one millimeter in diameter.A snap button can work for this purpose. Now add an infrared photo sensor to count the revolutions. First, incorporate the IR sensor into the circuit board, and then, screw the circuit board into the L plate.The path of the plastic part of the crossed needles must pass directly below the sensor for the assembly to function. Next, secure a 150 milliwatt infrared LED attached to circuit substrate to a small magnet using glue. Then place the LED on the base plate beneath the photo sensor.Finally, connect the photo sensor to an analog input channel of an AD convertor. Next bundle all the cabling and fix the cables to the plate. Lastly, connect the AD convertor to a personal computer via a USB cable.On the morning of the experiment collect all of the freshly emerged P.Quercivorus adults from a dead Qercus crispula bloom tree. More than 100 beetles are commonly harvested. Next, put a beetle on ice for anesthetization.Avoid getting the beetle wet, or it will be difficult to complete the procedure. Now, using two component glue, put a dab of the jelly consistency component on the tip of the mill arm. Then attach the mill arm to the beetle's pronotum.Next, using a fine needle, mix a small amount of the liquid glue component into a jelly glue component. Do not get any glue on the beetle's wings. Once the glue is hardened, use a magnet on the top plate to slide the plates apart and easily reinsert the flight mill into the apparatus.Next, tweak the position of the infrared LED so that it is directly beneath the sensor. When the beetle awakens it will begin to fly. Then start recording the amplified output from the photo sensor at a sampling rate of 1000 hertz.As the mill spins with the beetle's flight the IR beam gets disrupted by the plastic piece leading to a voltage jump on the sensor from 0 to 3.7 volts. If the recording threshold exceeds 0.5 volts, the data is good for analysis. Using the software controls start the recording by clicking on the plus symbol on the logging icon in the workspace window.Then right-click on the log name and select Begin Logging Set"To stop the recording, right-click the log name and select End Logging Set"The data will be saved to a csv file. In these experiment about half of the beetles attached to the flight mill showed one or more revolutions. Sometime after being attached most beetles started flying for a certain period and then flew again after a rest interval.Each beetle was recorded for at least one hour. The average beetle flew at 0.75 to 1.5 meters per second. Any beetle that flew less than a kilometer was omitted from the analysis.For 16 beetles that met this criterion the minimum flight time was 1.26 hours without energy intake, and the longest time and distance was 7.5 hours and 27.1 kilometers. In this cohort, no significant difference was found between males and females. After watching this video you will quickly collect plenty of data because this flight mill is low-cost, small, and easily constructed.And not to much many flight mill. Since its development, this method has paved the way for research in the field of basic biology as well as pest management to explore new avenues of the physiology and ecology of small flying insects. By modifying this method, for example by installing an odor delivery system or illumination system, we can explore how sensory information influence the flying capacity.

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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 ...

A Strain Gauge Monitor (SGM) for Continuous Valve Gape Measurements in Bivalve Molluscs in Response to Laboratory Induced Diel-cycling Hypoxia and pH

An inexpensive, laboratory-based, strain gauge valve gape monitor (SGM) was developed to monitor the valve gape behavior of bivalve molluscs in response to diel-cycling hypoxia. A Wheatstone bridge was connected to strain gauges that were attached to the shells of oysters (Crassostrea virginica). The recorded signals allowed for the opening and closing of the bivalves to be recorded continuously over two-day periods of experimentally-induced diel-cycling hypoxia and diel-cycling changes in pH. Here, we describe a protocol for developing an inexpensive strain gauge monitor and describe, in an example laboratory experiment, how we used it to measure the valve gape behavior of Eastern oysters (C. virginica), in response to diel-cycling hypoxia and cyclical changes in pH. Valve gape was measured on oysters subjected to cyclical severe hypoxic (0.6 mg/L) dissolved oxygen conditions with and without cyclical changes in pH, cyclical mild hypoxic (1.7 mg/L) conditions and normoxic (7.3 mg/L) conditions. We demonstrate that when oysters encounter repeated diel cycles, they rapidly close their shells in response to severe hypoxia and close with a time lag to mild hypoxia. When normoxia is restored, they rapidly open again. Oysters did not respond to cyclical pH conditions superimposed on diel cycling severe hypoxia. At reduced oxygen conditions, more than one third of the oysters closed simultaneously. We demonstrate that oysters respond to diel-cycling hypoxia, which must be considered when assessing the behavior of bivalves to dissolved oxygen. The valve SGM can be used to assess responses of bivalve molluscs to changes in dissolved oxygen or contaminants. Sealing techniques to better seal the valve gape strain gauges from sea water need further improvement to increase the longevity of the sensors.

A Strain Gauge Monitor SGM for Continuous Valve Gape Measurements in Bivalve Molluscs in Response to Laboratory Induced Diel-cycling Hypoxia and pH

Understanding the behavioral responses of bivalve suspension-feeders to environmental variables, such as dissolved oxygen, can explain some ecosystem processes. We have developed an inexpensive, laboratory-based, strain gauge monitor (SGM) to measure valve gape responses of oysters, Crassostrea virginica, to diel-cycling hypoxia and cyclical pH.

An inexpensive, laboratory-based, strain gauge valve gape monitor (SGM) was developed to monitor the valve gape behavior of bivalve molluscs in response ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Construction of a Compact Low-Cost Radiation Shield for Air-Temperature Sensors in Ecological Field Studies

Low cost temperature sensors are increasingly used by ecologists to assess climatic variation and change on ecologically relevant scales. Although cost-effective, if not deployed with proper solar radiation shielding, the observations recorded from these sensors will be biased and inaccurate. Manufactured radiation shields are effective at minimizing this bias, but are expensive compared to the cost of these sensors. Here, we provide a detailed methodology for constructing a compact version of a previously described custom fabricated radiation shield, which is more accurate than other published shielding methods that attempt to minimize shield size or construction costs. The method requires very little material: corrugated plastic sheets, aluminum foil duct tape, and cable ties. One 15 cm and two 10 cm squares of corrugated plastic are used for each shield. After cutting, scoring, taping and stapling of the sheets, the 10 cm squares form the bottom two layers of the solar radiation shield, while the 15 cm square forms the top layer. The three sheets are held together with cable ties. This compact solar radiation shield can be suspended, or placed against any flat surface. Care must be taken to ensure that the shield is completely parallel to the ground to prevent direct solar radiation from reaching the sensor, possibly causing increased warm biases in sun-exposed sites in the morning and afternoon relative to the original, larger design. Even so, differences in recorded temperatures between the smaller, compact shield design and the original design were small (mean daytime bias = 0.06 &#176;C). Construction costs are less than half of the original shield design, and the new design results in a less conspicuous instrument that may be advantageous in many field ecology settings.

With the advent of small, low-cost environmental sensors, it is now possible to deploy high-density networks of sensors to measure hyper localized temperature variation. Here, we provide a detailed methodology for constructing a compact version of a previously described custom-fabricated radiation shield for use with inexpensive thermochrons.

Low cost temperature sensors are increasingly used by ecologists to assess climatic variation and change on ecologically relevant scales. Although ...

Construction of a Low-cost Mobile Incubator for Field and Laboratory Use

Incubators are essential for a range of culture-based microbial methods, such as membrane filtration followed by cultivation for assessing drinking water quality. However, commercially available incubators are often costly, difficult to transport, not flexible in terms of volume, and/or poorly adapted to local field conditions where access to electricity is unreliable. The purpose of this study was to develop an adaptable, low-cost and transportable incubator that can be constructed using readily available components. The electronic core of the incubator was first developed. These components were then tested under a range of ambient temperature conditions (3.5 &#176;C - 39 &#176;C) using three types of incubator shells (polystyrene foam box, hard cooler box, and cardboard box covered with a survival blanket). The electronic core showed comparable performance to a standard laboratory incubator in terms of the time required to reach the set temperature, inner temperature stability and spatial dispersion, power consumption, and microbial growth. The incubator set-ups were also effective at moderate and low ambient temperatures (between 3.5 &#176;C and 27 &#176;C), and at high temperatures (39 &#176;C) when the incubator set temperature was higher. This incubator prototype is low-cost (&lt; 300 USD) and adaptable to a variety of materials and volumes. Its demountable structure makes it easy to transport. It can be used in both established laboratories with grid power or in remote settings powered by solar energy or a car battery. It is particularly useful as an equipment option for field laboratories in areas with limited access to resources for water quality monitoring.

This paper describes a method for building an adaptable, low-cost and transportable incubator for microbial testing of drinking water. Our design is based on widely available materials and can operate under a range of field conditions, while still offering the advantages of higher-end laboratory-based models.

Incubators are essential for a range of culture-based microbial methods, such as membrane filtration followed by cultivation for assessing drinking water ...

BtM, a Low-cost Open-source Datalogger to Estimate the Water Content of Nonvascular Cryptogams

Communities of nonvascular cryptogams, such as mosses or lichens, are an important part of the Earth's biodiversity, contributing to the regulation of the carbon and nitrogen cycles in many ecosystems. Being poikilohydric organisms, they do not actively control their internal water content and need a humid environment to activate their metabolism. Therefore, studying water relationships of nonvascular cryptogams is crucial to understand both their diversity patterns and their functions in the ecosystems. We present the BtM datalogger, a low-cost open-source platform for the study of the water content of nonvascular cryptogams. The datalogger is designed to measure ambient temperature, humidity, and conductance from up to eight samples simultaneously. We provide a design for a printed circuit board (PCB), a detailed protocol to assemble the components, and the required source code. All this makes the assembly of the BtM datalogger accessible to any research group, even to those without previous specialized knowledge. Therefore, the design presented here has the potential to help popularize the use of this type of device among ecologists and field biologists.

We present a simple and cost-effective method to build an open-source datalogger that measures the conductance of nonvascular cryptogams together with the environmental temperature and humidity. We describe the hardware design of the datalogger and provide step-by-step assembly instructions, the list of required open-source logging software, the code to run the datalogger, and a calibration protocol.

Communities of nonvascular cryptogams, such as mosses or lichens, are an important part of the Earth's biodiversity, contributing to the regulation of the ...